对相关申请的交叉引用Cross References to Related Applications
本申请要求2015年10月30日提交的美国临时专利申请号62/249,020和2015年11月4日提交的美国临时专利申请号62/250,965的优先权利益，所述两个临时专利申请都通过引用整体并入本文。This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/249,020, filed October 30, 2015, and U.S. Provisional Patent Application No. 62/250,965, filed November 4, 2015, both by This reference is incorporated herein in its entirety.
序列表sequence listing
本申请包含已经以ASCII格式电子提交的序列表，并且该序列表通过引用整体结合于此。所述ASCII副本创建于2016年10月27日，名称为P33044-WO.txt并且大小为172,164字节。This application contains a Sequence Listing that has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy was created on October 27, 2016, named P33044-WO.txt and was 172,164 bytes in size.
背景技术Background technique
治疗性抗体的开发代表着人类医药的悠久历史中的革命性纪元。超过30种抗体已被批准用于人类治疗，并且在世界范围内超过250种抗体处于临床开发阶段以用于多种重大疾病，包括癌症、自身免疫、炎症、心血管病、传染病和眼病。在过去十年，单克隆抗体产品的市场呈指数增长，其受轰动性药物如曲妥珠单抗、贝伐单抗、利妥昔单抗、英夫利昔单抗和阿达木单抗的成功的推进。这些第一代抗体治疗剂已经使众多患者受益，同时抗体技术的进步和对作用机制的更深理解为具有甚至更佳效力和更少副作用的改进版本的抗体铺平了道路。The development of therapeutic antibodies represents a revolutionary era in the long history of human medicine. More than 30 antibodies have been approved for human therapy, and more than 250 antibodies are in clinical development worldwide for a variety of major diseases, including cancer, autoimmunity, inflammation, cardiovascular disease, infectious disease and eye disease. The market for monoclonal antibody products has grown exponentially over the past decade, driven by the success of blockbuster drugs such as trastuzumab, bevacizumab, rituximab, infliximab and adalimumab advance. These first-generation antibody therapeutics have benefited numerous patients, while advances in antibody technology and a better understanding of the mechanism of action have paved the way for improved versions of antibodies with even greater potency and fewer side effects.
与作为小的有机和无机分子的常规药物相比，抗体治疗剂的成功开发和可行的利用具有许多独特的挑战。与所有蛋白质一样，抗体的生物物理性质对于其行为是重要的决定因素并且显著影响涉及表达、纯化、制剂、存储、递送、药物动力学、免疫原性和用药方案的治疗剂的开发。在众多性质中，蛋白质稳定性是决定候选抗体品质及其作为成功治疗剂的合意性的主要特征。The successful development and viable utilization of antibody therapeutics presents many unique challenges compared to conventional drugs, which are small organic and inorganic molecules. As with all proteins, the biophysical properties of antibodies are important determinants of their behavior and significantly affect the development of therapeutics involving expression, purification, formulation, storage, delivery, pharmacokinetics, immunogenicity, and dosing regimens. Amongst many properties, protein stability is the main feature that determines the quality of an antibody candidate and its desirability as a successful therapeutic.
蛋白质疗法通常需要将高剂量的蛋白质递送至患者以便实现所需的效力。同时，某些给药途径与限制如递送时间、体积和体力有关，其需要高剂量蛋白质在高浓度制剂(例如，至少100mg/ml)中。然而，高浓度蛋白质制剂提出了关于稳定性、溶解性、粘性及其他蛋白质性质的特别挑战。Protein therapy often requires the delivery of high doses of protein to the patient in order to achieve the desired efficacy. At the same time, certain routes of administration are associated with limitations such as delivery time, volume, and physical effort, which require high doses of protein in high concentration formulations (eg, at least 100 mg/ml). However, high concentration protein formulations present special challenges regarding stability, solubility, viscosity, and other protein properties.
蛋白质可以是不稳定的并且经由多种物理和化学分解途径而被分解。物理不稳定性主要经由两种途径发生–变性和聚集，而化学不稳定性可以经由多种途径发生，如脱酰胺、异构化、交联、氧化和片段化。抗体不稳定性对于药物开发是不利的，因为其可能导致活性药物量的减小和较低的体内效力，治疗剂的各批次间的可变性增加，并且可能是最重要的，患者中针对聚集物和降解物的免疫原性。Wang等(2007)J.Pharm.Sci.96:1-26；Moore等(1980)J Clin Endocrinology&Metabolism 51:691-697；Rosenberg等(2006)AAPSJ 8:E501-7；Joubert等(2011)J Biol Chem 286:25118-25133；Joubert等(2012)J Biol Chem(2012)286:25266-79)。Proteins can be unstable and break down via a variety of physical and chemical breakdown pathways. Physical instability occurs mainly through two pathways – denaturation and aggregation, while chemical instability can occur through multiple pathways such as deamidation, isomerization, cross-linking, oxidation and fragmentation. Antibody instability is detrimental to drug development as it can lead to reduced amounts of active drug and lower in vivo potency, increased batch-to-batch variability of therapeutics and, perhaps most importantly, patient targeting Immunogenicity of aggregates and degradants. Wang et al. (2007) J.Pharm.Sci.96:1-26; Moore et al. (1980) J Clin Endocrinology & Metabolism 51:691-697; Rosenberg et al. (2006) AAPSJ 8:E501-7; Joubert et al. (2011) J Biol Chem 286:25118-25133; Joubert et al. (2012) J Biol Chem (2012) 286:25266-79).
抗体是大的多结构域蛋白质，并且对其稳定性和聚集倾向性有作用的因素是复杂的，包括许多外在条件如温度、pH、浓度、离子强度和物理应力。同样重要的是蛋白质本身的一级序列。虽然在特定同种型的抗体之间Fc区域的性质在很大程度上相同，但是Fab区有很大不同。因此，很大程度上由于Fab序列差异和抗体的特别的抗原特异性，在抗体之间在稳定性和聚集倾向方面存在显著差异。Lowe等(2011)Adv.Protein Chem.Struct Biol.84:41-61。Antibodies are large multi-domain proteins, and the factors that contribute to their stability and aggregation propensity are complex, including many extrinsic conditions such as temperature, pH, concentration, ionic strength, and physical stress. Also important is the primary sequence of the protein itself. While the nature of the Fc region is largely the same between antibodies of a particular isotype, the Fab region is quite different. Thus, there are significant differences between antibodies in terms of stability and aggregation propensity, largely due to Fab sequence differences and the particular antigen specificity of the antibodies. Lowe et al. (2011) Adv. Protein Chem. Struct Biol. 84:41-61.
补体系统在免疫复合物的清除和对传染剂、外部抗原、病毒感染的细胞和肿瘤细胞的免疫反应方面发挥中心作用。然而，补体也参与病理炎症和自体免疫病。因此，抑制过度的或不受控的补体级联激活可以为患有此种疾病和病症的患者提供临床益处。The complement system plays a central role in the clearance of immune complexes and the immune response to infectious agents, foreign antigens, virus-infected cells and tumor cells. However, complement is also involved in pathological inflammation and autoimmune diseases. Therefore, inhibiting excessive or uncontrolled complement cascade activation may provide clinical benefit to patients suffering from such diseases and disorders.
补体系统包括三种不同的激活途径，其被指定为经典途径、结合甘露糖的凝集素途径和旁路途径。V.M.Holers于Clinical Immunology:Principles and Practice,ed.R.R.Rich,Mosby Press；1996,363-391。经典途径是钙/镁依赖性级联，其通常通过形成抗原-抗体复合物而激活。结合甘露糖的凝集素(MBL)途径通过MBL结合病原体上的糖类结构而引发，导致切割C2和C4以形成活性C2a、C2b、C4a和C4b的MBL蛋白酶(MASP)的活化。旁路途径是镁依赖性级联，其通过C3在某些易感表面(例如酵母和细菌的细胞壁多糖，和某些生物聚合材料)上的沉积和活化而激活。补体途径的激活产生补体蛋白质的生物活性片段，例如C3a、C4a和C5a过敏毒素和C5b-9膜攻击复合物(MAC)，其介导涉及白细胞趋化性、巨噬细胞、嗜中性粒细胞、血小板、肥大细胞和内皮细胞的激活、血管通透性、细胞溶解和组织损伤的炎性活动。The complement system includes three distinct pathways of activation designated as the classical pathway, the mannose-binding lectin pathway, and the alternative pathway. V.M. Holers in Clinical Immunology: Principles and Practice, ed. R.R. Rich, Mosby Press; 1996, 363-391. The classical pathway is a calcium/magnesium-dependent cascade that is usually activated by the formation of antigen-antibody complexes. The mannose-binding lectin (MBL) pathway is initiated by MBL binding to carbohydrate structures on pathogens, resulting in the activation of MBL protease (MASP) that cleaves C2 and C4 to form active C2a, C2b, C4a, and C4b. The alternative pathway is a magnesium-dependent cascade activated by the deposition and activation of C3 on certain susceptible surfaces such as cell wall polysaccharides of yeast and bacteria, and certain biopolymeric materials. Activation of the complement pathway produces biologically active fragments of complement proteins such as C3a, C4a, and C5a anaphylatoxins and the C5b-9 membrane attack complex (MAC), which mediates leukocyte chemotaxis, macrophages, neutrophils , activation of platelets, mast cells and endothelial cells, vascular permeability, cell lysis and inflammatory activity of tissue damage.
因子D是对于备选补体途径的激活必要的高度特异性的丝氨酸蛋白酶。其切割与C3b结合的因子B，产生作为旁路途径C3/C5转化酶的活性组分的C3b/Bb酶。因子D可以作为抑制的合适靶标，因为其在人中的血浆浓度非常低(1.8μg/ml)，并且已经证明其是旁路补体途径的激活的限速酶。P.H.Lesavre和H.J.Müller-Eberhard.(1978)J.Exp.Med.148:1498-1510；J.E.Volanakis等人(1985)New Eng.J.Med.312:395-401。Factor D is a highly specific serine protease essential for the activation of the alternative complement pathway. It cleaves Factor B bound to C3b, yielding the C3b/Bb enzyme that is the active component of the alternative pathway C3/C5 convertase. Factor D could be a suitable target for inhibition because its plasma concentration in humans is very low (1.8 μg/ml) and it has been shown to be the rate-limiting enzyme for activation of the alternative complement pathway. P. H. Lesavre and H. J. Müller-Eberhard. (1978) J. Exp. Med. 148:1498-1510; J. E. Volanakis et al. (1985) New Eng. J. Med. 312:395-401.
已经证明，在动物模型中以及在离体研究中，补体激活的下调在治疗若干疾病适应证方面是有效的，所述疾病适应证例如是系统性红斑狼疮和肾小球肾炎、类风湿性关节炎、心肺分流术(cardiopulmonary bypass)和血液透析、器官移植中的超急性排斥、心肌梗死、再灌注损伤和成人呼吸窘迫综合征。另外，其它炎性病症和自体免疫/免疫复合物疾病也与补体激活密切相关，所述疾病包括热损伤、严重哮喘、过敏性休克、肠炎、荨麻疹、血管性水肿、血管炎、多发性硬化、重症肌无力、膜性增生性肾小球肾炎和舍格伦综合征。Downregulation of complement activation has been shown to be effective in the treatment of several disease indications in animal models as well as in ex vivo studies, such as systemic lupus erythematosus and glomerulonephritis, rheumatoid arthritis inflammation, cardiopulmonary bypass and hemodialysis, hyperacute rejection in organ transplantation, myocardial infarction, reperfusion injury, and adult respiratory distress syndrome. In addition, other inflammatory conditions and autoimmune/immune complex diseases are also strongly associated with complement activation, including heat injury, severe asthma, anaphylactic shock, enteritis, urticaria, angioedema, vasculitis, multiple sclerosis , myasthenia gravis, membranous proliferative glomerulonephritis, and Sjögren syndrome.
年龄相关性黄斑变性(AMD)是中央视网膜的进行性的慢性疾病，其对视觉敏锐性有显著后果。Lim等人(2012)Lancet 379:1728。在工业化国家中，该疾病的晚期形式是视力丧失的主要原因。对于≥40岁的高加索人群，早期AMD的患病率估计在6.8％而晚期AMD在1.5％。de Jong(2006)N.Engl.J.Med.355:1474。在80岁后，晚期AMD的患病率随年龄增长显著增加至11.8％。存在两种类型的AMD，非渗出性(干性)AMD和渗出性(湿性)AMD。更常见的干性形式AMD包括中央视网膜(黄斑)下的视网膜色素上皮(RPE)的萎缩和肥大性改变以及RPE上的沉积物(玻璃疣)。晚期干性AMD可以导致显著的视网膜损伤，包括地图状萎缩(GA)，以及不可逆的视力丧失。此外，患有干性AMD的患者可以进展至湿性形式，其中在视网膜下发展出被称为脉络膜新生血管膜(CNVM)的异常血管，渗液和渗血，并且最终导致视网膜中和视网膜下的致盲性盘状瘢痕。Age-related macular degeneration (AMD) is a progressive, chronic disease of the central retina with dramatic consequences on visual acuity. Lim et al. (2012) Lancet 379:1728. Advanced forms of the disease are the leading cause of vision loss in industrialized countries. For the Caucasian population ≥40 years of age, the prevalence of early AMD is estimated at 6.8% and late AMD at 1.5%. de Jong (2006) N. Engl. J. Med. 355:1474. After the age of 80 years, the prevalence of advanced AMD increased significantly with age to 11.8%. There are two types of AMD, non-exudative (dry) AMD and exudative (wet) AMD. The more common dry form of AMD involves atrophic and hypertrophic changes in the retinal pigment epithelium (RPE) beneath the central retina (macula) and deposits (drusen) on the RPE. Advanced dry AMD can lead to significant retinal damage, including geographic atrophy (GA), and irreversible vision loss. In addition, patients with dry AMD can progress to the wet form, in which abnormal blood vessels called choroidal neovascular membrane (CNVM) develop under the retina, ooze and bleed, and eventually lead to Blinding discoid scar.
靶向新血管形成(新血管生成)的药物已经是治疗湿性AMD的主要依靠。雷珠单抗(Ranibizumab)，一种抗-VEGFA抗体片段，已被证明可以高效地改善患有湿性AMD的患者的视力。最近的研究暗示了AMD与补体级联中的关键蛋白质之间的关联性，并且正在开发多种靶向特定补体组分的疗法用以治疗干性AMD。通过结合因子D上的外部位点而有效抑制因子D及补体旁路途径的人源化抗-因子D Fab片段(aFD，lampalizumab；FCFD4514S)目前处于临床开发中以用于治疗与干性AMD相关的GA。Katschke等人(2012)J.Biol.Chem.287:12886。最近的II期临床试验显示每月玻璃体内注射lampalizumab有效地减慢患有晚期干性AMD的患者中的GA病变的进展。Drugs that target the formation of new blood vessels (neovascularization) have been the mainstay of treatment for wet AMD. Ranibizumab, an anti-VEGFA antibody fragment, has been shown to be highly effective in improving vision in patients with wet AMD. Recent studies have implicated a link between AMD and key proteins in the complement cascade, and several therapies targeting specific complement components are being developed for the treatment of dry AMD. A humanized anti-factor D Fab fragment (aFD, lampalizumab; FCFD4514S), which potently inhibits factor D and the alternative complement pathway by binding to an exosite on factor D, is currently in clinical development for the treatment of dry AMD associated the ga. Katschke et al. (2012) J. Biol. Chem. 287:12886. A recent phase II clinical trial showed monthly intravitreal injections of lampalizumab effectively slowed the progression of GA lesions in patients with advanced dry AMD.
眼睛具有许多独特的生物物理和解剖学特征，其使得眼部药物递送更具有挑战性。例如，血-眼屏障是保护眼睛免于感染的防御机制，但是同时使得药物难于渗入，尤其是对于眼睛后段中的疾病。因此，通常需要高剂量施用来实现和保持药物的原位生物利用度(例如，眼部停留时间)以便改善效力。同时，眼睛后部中的有限空间限制了递送的药物体积，其又要求药物在高浓度制剂中递送。The eye has many unique biophysical and anatomical features that make ocular drug delivery more challenging. For example, the blood-ocular barrier is a defense mechanism that protects the eye from infection, but at the same time makes it difficult for drugs to penetrate, especially for diseases in the posterior segment of the eye. Therefore, high dose administration is often required to achieve and maintain in situ bioavailability (eg, ocular residence time) of the drug in order to improve efficacy. At the same time, the limited space in the back of the eye limits the volume of drug delivered, which in turn requires the drug to be delivered in a high concentration formulation.
患有眼病的患者也可以受益于治疗剂的长效/缓释递送。较低频率的给药将为患者提供改善的便利性，具有降低感染率和增加临床效力的潜在益处。高剂量药物的受控释放还可以使药物副作用最小化。两种有希望的用于长效递送的系统是基于PLGA的固体植入物和可植入端口递送系统(PDS)。两种系统都有可能提供接近零级的释放动力学用于延长的时间。对于PLGA植入物，蛋白质药物被包封在疏水聚合物基质中并且药物释放经由聚合物的缓慢水解实现。释放速率可以通过改变药物载量、聚合物疏水性或聚合物分子量来控制。PDS是一种可再填充的装置，其中向玻璃体内的释放通过包含钛玻璃料的多孔金属膜来控制。因为储库具有小的体积，所以利用PDS进行有效递送需要高的蛋白浓度。Patients with eye diseases may also benefit from long-acting/sustained-release delivery of therapeutic agents. Less frequent dosing would provide improved convenience to patients, with potential benefits of reduced infection rates and increased clinical efficacy. Controlled release of high doses of drugs can also minimize drug side effects. Two promising systems for long-term delivery are PLGA-based solid implants and implantable port delivery systems (PDS). Both systems have the potential to provide near zero order release kinetics for extended periods of time. For PLGA implants, protein drugs are encapsulated in a hydrophobic polymer matrix and drug release is achieved via slow hydrolysis of the polymer. The rate of release can be controlled by varying the drug loading, polymer hydrophobicity, or polymer molecular weight. The PDS is a refillable device in which release into the vitreous is controlled by a porous metal membrane containing titanium frit. Efficient delivery using PDS requires high protein concentrations because of the small volume of the depot.
药物所暴露的条件根据使用的递送系统而变化。对于在固体PLGA植入物中的结合，使用冻干的或喷雾干燥的药物。使用热熔挤出法来制备植入物，使得药物短暂地暴露于接近90℃的温度。虽然药物在释放过程中保持固态，但是PLGA的降解可以将药物暴露于低pH环境。相反，利用PDS递送的药物被保持在液态下的高浓度并被暴露于玻璃体，玻璃体被表征为一种具有减小的在生理离子强度和pH下还原的环境。The conditions to which the drug is exposed will vary depending on the delivery system used. For incorporation in solid PLGA implants, lyophilized or spray-dried drugs were used. Implants were prepared using a hot-melt extrusion method, which briefly exposed the drug to temperatures close to 90°C. Although the drug remains solid during release, the degradation of PLGA can expose the drug to a low pH environment. In contrast, drugs delivered using PDS are maintained at high concentrations in the liquid state and exposed to the vitreous, which is characterized as an environment with reduced reduction at physiological ionic strength and pH.
除了高浓度和长效递送以外或作为高浓度和长效递送的替代，药物的增加的生物利用度(例如，眼部停留时间)可以通过翻译后修饰实现或受其促进，其中蛋白质药物与天然的或合成的聚合物共价缀合，如聚唾液酸化、HES化(与羟乙基淀粉缀合)和PEG化。Chen等人(2011)Expert.Opin.Drug Deliv.8:1221-36；Kontermann(2009)BioDrugs 23:93-109。PEG化，即将聚合物聚乙二醇(PEG)共价连接至蛋白质，是一种尤其可用于延长抗体片段治疗剂的半衰期的确立已久的技术。Jevsevar等人(2010)Biotech.J.5:113-128。In addition to or as an alternative to high-concentration and long-acting delivery, increased bioavailability (e.g., ocular residence time) of a drug can be achieved or facilitated by post-translational modifications, where protein drugs are similar to natural Covalently conjugated or synthetic polymers such as polysialylation, HESylation (conjugation to hydroxyethyl starch) and PEGylation. Chen et al. (2011) Expert. Opin. Drug Deliv. 8:1221-36; Kontermann (2009) BioDrugs 23:93-109. PEGylation, the covalent attachment of the polymer polyethylene glycol (PEG) to proteins, is a well-established technique that is particularly useful for extending the half-life of antibody fragment therapeutics. Jevsevar et al. (2010) Biotech. J. 5:113-128.
因此，对于具有改善的稳定性以及在某些实施方案中适合于高浓度制剂和/或长效递送的抗-因子D抗体及其缀合物存在强烈的需求。Accordingly, there is a strong need for anti-Factor D antibodies and conjugates thereof that have improved stability and, in certain embodiments, are suitable for high concentration formulations and/or long-term delivery.
发明概述Summary of the invention
本发明部分基于这样的发现，即抗体中对鉴定的热点的靶向氨基酸置换可以有效地提高抗体的稳定性和作为治疗剂的整体效力。如与未缀合的抗体相比，此种抗体与多臂聚合物(诸如多臂多元醇)的缀合可以提高玻璃体液半衰期、房水半衰期和/或视网膜半衰期。The present invention is based in part on the discovery that targeted amino acid substitutions in antibodies to identified hotspots can be effective in improving antibody stability and overall efficacy as therapeutics. Conjugation of such antibodies to multi-armed polymers, such as multi-armed polyols, can increase vitreous humor half-life, aqueous humor half-life, and/or retinal half-life as compared to unconjugated antibodies.
在一些方面，本发明涉及包含与一种或多种多臂多元醇共价连接的一种或多种抗-因子D抗体或抗-因子D抗体变体的缀合物。在某些实施方案中，多元醇是八臂多元醇(即，八聚体)。在一些实施方案中，多元醇是聚乙二醇(PEG)。在某些实施方案中，PEG可以具有通式(Ia)、(Ib)、(IIa)、(IIIa)或(IVa)中任一种的结构(如下文所示)。In some aspects, the invention relates to conjugates comprising one or more anti-Factor D antibodies or anti-Factor D antibody variants covalently linked to one or more multi-armed polyols. In certain embodiments, the polyol is an eight-armed polyol (ie, an octamer). In some embodiments, the polyol is polyethylene glycol (PEG). In certain embodiments, PEG can have the structure of any of the general formulas (Ia), (Ib), (IIa), (Ilia) or (IVa) (shown below).
用于本发明的缀合物中的抗-因子D抗体变体具有改善的稳定性。抗-因子D抗体变体包含在参比抗-因子D抗体的高变区(HVR)内的至少一个靶天冬氨酸(D或Asp)残基的置换，其中所述靶Asp残基被鉴定为倾向于异构化并且所述置换是Asp至谷氨酸(E或Glu)，并且其中当与所述参比抗-因子D抗体相比时，所述抗-因子D抗体变体显示改善的稳定性而不显著丧失因子D结合亲和力。在一些方面，进行置换的靶Asp残基在Asp-Xaa基序内，其中Xaa是Asp、Gly、His、Ser或Thr。在一些方面，靶Asp残基是Asp-Asp(DD)基序中的第一个Asp。在一些方面，抗-因子D抗体变体包含在参比抗-因子D抗体的HVR内的另外的Asp位点处的一个或多个置换，其中所述置换是Asp至丝氨酸(S或Ser)以减少抗体的整体电荷，由此提高抗体的溶解性。在一些方面，抗-因子D抗体变体包含在被鉴定为倾向于脱酰胺的天冬酰胺(N或Asn)位点处的一个或多个置换，其中所述置换是Asn至Ser以减少或消除抗体的脱酰胺。The anti-Factor D antibody variants used in the conjugates of the invention have improved stability. Anti-Factor D antibody variants comprise a substitution of at least one target Aspartate (D or Asp) residue within the hypervariable region (HVR) of a reference anti-Factor D antibody, wherein the target Asp residue is replaced by Identified as prone to isomerization and said substitution is Asp to glutamic acid (E or Glu), and wherein said anti-Factor D antibody variant shows when compared to said reference anti-Factor D antibody Improved stability without significant loss of Factor D binding affinity. In some aspects, the Asp residue targeted for substitution is within the Asp-Xaa motif, where Xaa is Asp, Gly, His, Ser, or Thr. In some aspects, the target Asp residue is the first Asp in the Asp-Asp(DD) motif. In some aspects, the anti-Factor D antibody variant comprises one or more substitutions at an additional Asp site within the HVR of the reference anti-Factor D antibody, wherein the substitution is Asp to Serine (S or Ser) To reduce the overall charge of the antibody, thereby improving the solubility of the antibody. In some aspects, the anti-Factor D antibody variant comprises one or more substitutions at an asparagine (N or Asn) site identified as prone to deamidation, wherein the substitution is Asn to Ser to reduce or Eliminates deamidation of antibodies.
在一些实施方案中，抗-因子D抗体变体是Fab片段，其中所述Fab片段的重链的C-末端以氨基酸“CDKTHT”、“CDKTHL”、“CDKTH”、“CDKT”、“CDK”或“CD”结束。在一些实施方案中，Fab片段的重链的C-末端以序列“CDKTHX”结束，其中X是除T以外的任何氨基酸。C-末端处的截短和/或突变能够减少或消除针对Fab的AHA反应性，而不危害热稳定性或表达。在一些实施方案中，Fab片段的重链的C-末端以氨基酸“CDKTHTC”、“CDKTHTCPPC”、“CDKTHTCPPS”、“CDKTHTSPPC”、“CDKTHTAPPC”、“CDKTHTSGGC”或“CYGPPC”结束。在一些这样的实施方案中，C-末端氨基酸中的游离半胱氨酸可以接受缀合至例如聚合物(诸如PEG)。在一些实施方案中，Fab片段包含选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ IDNO:54(以“CDKTHT”结束)、55-66(以“CDKTHL”、“CDKTHTC”、“CPPC”、“CPPS”、“SPPC”、“APPC”、“SGGC”、“CYGPPC”、“CDKTH”、“CDKT”、“CDK”或“CD”结束)、和116(以“CDKTHX”结束)。在一些实施方案中，Fab是包含SEQ ID NO:67(以“VERK”结束)的重链恒定结构域氨基酸序列的IgG2Fab片段，或包含SEQ ID NO:68(以“VERKC”结束)的重链恒定结构域氨基酸序列的IgG2Fab-C片段。在一些实施方案中，Fab是包含选自由以下组成的组的重链恒定结构域氨基酸序列的IgG4Fab片段：SEQ ID NO:69-73(以“KYGPP”、“KYGP”、“KYG”、“KY”或“K”结束)，或包含SEQ ID NO:74(以“KYGPPC”结束)的重链恒定结构域氨基酸序列的IgG4Fab-C片段。作为C末端处的截短和/或突变的替代方式，为了避免预先存在的抗铰链抗体(PE-AHA)应答，可以使用IgG2或IgG4Fab片段，因为这些片段不显示PE-AHA应答。In some embodiments, the anti-Factor D antibody variant is a Fab fragment, wherein the C-terminus of the heavy chain of the Fab fragment is terminated by the amino acids "CDKTHT", "CDKTHL", "CDKTH", "CDKT", "CDK" or "CD" ends. In some embodiments, the C-terminus of the heavy chain of the Fab fragment ends with the sequence "CDKTHX", where X is any amino acid except T. Truncation and/or mutation at the C-terminus can reduce or eliminate AHA reactivity towards the Fab without compromising thermostability or expression. In some embodiments, the C-terminus of the heavy chain of the Fab fragment ends with the amino acid "CDKTHTC", "CDKTHTCPPC", "CDKTHTCPPS", "CDKTHTSPPC", "CDKTHTAPPC", "CDKTHTSGGC", or "CYGPPC". In some such embodiments, the free cysteine in the C-terminal amino acid is receptive to conjugation to, for example, a polymer such as PEG. In some embodiments, the Fab fragment comprises a heavy chain constant domain amino acid sequence selected from the group consisting of: SEQ ID NO:54 (ending with "CDKTHT"), 55-66 (ending with "CDKTHL", "CDKTHTC", " CPPC", "CPPS", "SPPC", "APPC", "SGGC", "CYGPPC", "CDKTH", "CDKT", "CDK", or "CD"), and 116 (ending with "CDKTHX") . In some embodiments, the Fab is an IgG2 Fab fragment comprising the heavy chain constant domain amino acid sequence of SEQ ID NO: 67 (ending in "VERK"), or comprising the heavy chain of SEQ ID NO: 68 (ending in "VERKC") IgG2 Fab-C fragment of constant domain amino acid sequence. In some embodiments, the Fab is an IgG4 Fab fragment comprising a heavy chain constant domain amino acid sequence selected from the group consisting of: SEQ ID NOs: 69-73 (identified as "KYGPP", "KYGP", "KYG", "KY " or "K" ending), or an IgG4 Fab-C fragment comprising the heavy chain constant domain amino acid sequence of SEQ ID NO: 74 (ending with "KYGPPC"). As an alternative to truncation and/or mutation at the C-terminus, to avoid a pre-existing anti-hinge antibody (PE-AHA) response, IgG2 or IgG4 Fab fragments can be used, as these fragments do not show a PE-AHA response.
在一些方面，用于产生用于本发明的缀合物的抗体变体的参比抗-因子D抗体包含SEQ ID NO:3的轻链可变结构域序列，SEQ ID NO:4的重链可变结构域序列，或两者。随后，所得的抗体变体可以包含SEQ ID NO:11的轻链HVR1(HVR-L1)序列和SEQ ID NO:12的重链HVR2(HVR-H2)序列，或可以包含SEQ ID NO:13的轻链HVR3(HVR-L3)序列，或可以包含SEQID NO:14的轻链HVR1(HVR-L1)序列和SEQ ID NO:12的重链HVR2(HVR-H2)序列，或可以包含SEQ ID NO:15的重链HVR3(HVR-H3)序列。In some aspects, the reference anti-Factor D antibody used to generate the antibody variants for the conjugates of the invention comprises the light chain variable domain sequence of SEQ ID NO:3, the heavy chain of SEQ ID NO:4 variable domain sequences, or both. Subsequently, the resulting antibody variant may comprise the light chain HVR1 (HVR-L1) sequence of SEQ ID NO: 11 and the heavy chain HVR2 (HVR-H2) sequence of SEQ ID NO: 12, or may comprise the sequence of SEQ ID NO: 13 The light chain HVR3 (HVR-L3) sequence, or may comprise the light chain HVR1 (HVR-L1) sequence of SEQ ID NO: 14 and the heavy chain HVR2 (HVR-H2) sequence of SEQ ID NO: 12, or may comprise SEQ ID NO :15 heavy chain HVR3 (HVR-H3) sequence.
在一些方面，用于本发明的缀合物的抗-因子D抗体变体是参比抗-因子D抗体的变体，其中所述参比抗-因子D抗体包含SEQ ID NO:1的轻链序列和SEQ ID NO:2的重链序列，并且其中所述变体相对于参比抗-因子D抗体包含以下序列修饰：SEQ ID NO:11的轻链HVR1(HVR-L1)序列和SEQ ID NO:12的重链HVR2(HVR-H2)序列。此种变体在本文以下的实施例中被称为“TM”变体(AFD.v6)(参见，例如，表1)。In some aspects, the anti-Factor D antibody variant used in the conjugates of the invention is a variant of a reference anti-Factor D antibody, wherein the reference anti-Factor D antibody comprises the light of SEQ ID NO: 1 chain sequence and the heavy chain sequence of SEQ ID NO:2, and wherein the variant comprises the following sequence modifications relative to the reference anti-Factor D antibody: light chain HVR1 (HVR-L1) sequence of SEQ ID NO:11 and SEQ ID NO:11 Heavy chain HVR2 (HVR-H2) sequence of ID NO:12. This variant is referred to as the "TM" variant (AFD.v6) in the Examples herein below (see, eg, Table 1).
在一些方面，用于本发明的缀合物的抗-因子D抗体变体是参比抗-因子D抗体的变体，其中所述参比抗-因子D抗体包含SEQ ID NO:1的轻链序列和选自由以下组成的组的重链序列：SEQ ID NO:34-53和115，并且其中所述变体相对于参比抗-因子D抗体包含以下序列修饰：SEQ ID NO:11的轻链HVR1(HVR-L1)序列和SEQ ID NO:12的重链HVR2(HVR-H2)序列。此种变体被称为“修饰的TM”变体。这些修饰的TM变体包含不同于TM变体的重链恒定结构域，并且其选自由以下组成的组：SEQ ID NO:55-74和116。In some aspects, the anti-Factor D antibody variant used in the conjugates of the invention is a variant of a reference anti-Factor D antibody, wherein the reference anti-Factor D antibody comprises the light of SEQ ID NO: 1 chain sequence and a heavy chain sequence selected from the group consisting of: SEQ ID NO:34-53 and 115, and wherein the variant comprises the following sequence modifications relative to the reference anti-factor D antibody: SEQ ID NO:11 Light chain HVR1 (HVR-L1 ) sequence and heavy chain HVR2 (HVR-H2) sequence of SEQ ID NO:12. Such variants are referred to as "modified TM" variants. These modified TM variants comprise a heavy chain constant domain that differs from the TM variant and is selected from the group consisting of SEQ ID NOs: 55-74 and 116.
在一些方面，用于本发明的缀合物的抗-因子D抗体变体是参比抗-因子D抗体的变体，其中所述参比抗-因子D抗体包含SEQ ID NO:1的轻链序列和SEQ ID NO:2的重链序列，并且其中所述变体相对于参比抗-因子D抗体包含以下序列修饰：SEQ ID NO:11的轻链HVR1(HVR-L1)序列、SEQ ID NO:12的重链HVR2(HVR-H2)序列和SEQ ID NO:13的轻链HVR3(HVR-L3)序列。此种变体在本文以下的实施例中被称为“TM.D92E”变体(AFD.v7)(参见，例如，表1)。In some aspects, the anti-Factor D antibody variant used in the conjugates of the invention is a variant of a reference anti-Factor D antibody, wherein the reference anti-Factor D antibody comprises the light of SEQ ID NO: 1 chain sequence and the heavy chain sequence of SEQ ID NO:2, and wherein the variant comprises the following sequence modifications relative to the reference anti-Factor D antibody: light chain HVR1 (HVR-L1) sequence of SEQ ID NO:11, SEQ ID NO:11 The heavy chain HVR2 (HVR-H2) sequence of ID NO:12 and the light chain HVR3 (HVR-L3) sequence of SEQ ID NO:13. This variant is referred to as the "TM.D92E" variant (AFD.v7) in the Examples herein below (see, eg, Table 1).
在一些方面，用于本发明的缀合物的抗-因子D抗体变体是参比抗-因子D抗体的变体，其中所述参比抗-因子D抗体包含SEQ ID NO:1的轻链序列和选自由以下组成的组的重链序列：SEQ ID NO:34-53和115，并且其中所述变体相对于参比抗-因子D抗体包含以下序列修饰：SEQ ID NO:11的轻链HVR1(HVR-L1)序列、SEQ ID NO:12的重链HVR2(HVR-H2)序列和SEQ ID NO:13的轻链HVR3(HVR-L3)序列。此种变体被称为“修饰的TM.D92E”变体。这些修饰的TM.D92E变体包含不同于TM.D92E变体的重链恒定结构域，并且其选自由以下组成的组：SEQ ID NO:55-74和116。In some aspects, the anti-Factor D antibody variant used in the conjugates of the invention is a variant of a reference anti-Factor D antibody, wherein the reference anti-Factor D antibody comprises the light of SEQ ID NO: 1 chain sequence and a heavy chain sequence selected from the group consisting of: SEQ ID NO:34-53 and 115, and wherein the variant comprises the following sequence modifications relative to the reference anti-factor D antibody: SEQ ID NO:11 Light chain HVR1 (HVR-L1 ) sequence, heavy chain HVR2 (HVR-H2) sequence of SEQ ID NO:12, and light chain HVR3 (HVR-L3) sequence of SEQ ID NO:13. Such variants are referred to as "modified TM.D92E" variants. These modified TM.D92E variants comprise a heavy chain constant domain that differs from the TM.D92E variant and is selected from the group consisting of SEQ ID NOs: 55-74 and 116.
在一些方面，用于本发明的缀合物的抗-因子D抗体变体是参比抗-因子D抗体的变体，其中所述参比抗-因子D抗体包含SEQ ID NO:1的轻链序列和SEQ ID NO:2的重链序列，并且其中所述变体相对于参比抗-因子D抗体包含以下序列修饰：SEQ ID NO:14的轻链HVR1(HVR-L1)序列和SEQ ID NO:12的重链HVR2(HVR-H2)序列。此种变体在本文以下的实施例中被称为“SIESD”变体(AFD.v8)(参见，例如，表1)。在一些实施方案中，“SIESD”变体(AFD.v8)包含SEQ ID NO:26的轻链序列和SEQ ID NO:27的重链序列。在一些实施方案中，Cys修饰版本的“SIESD”变体包含SEQ ID NO:26的轻链序列和SEQ ID NO:30的重链序列。在一些实施方案中，Cys-Pro-Pro-Cys修饰版本的“SIESD”变体包含SEQ ID NO:26的轻链序列和SEQ IDNO:31的重链序列。在一些实施方案中，修饰版本的“SIESD”变体包含SEQ ID NO:26的轻链序列和选自由以下组成的组的重链序列：SEQ ID NO:75-92和117。In some aspects, the anti-Factor D antibody variant used in the conjugates of the invention is a variant of a reference anti-Factor D antibody, wherein the reference anti-Factor D antibody comprises the light of SEQ ID NO: 1 chain sequence and the heavy chain sequence of SEQ ID NO:2, and wherein the variant comprises the following sequence modifications relative to the reference anti-Factor D antibody: light chain HVR1 (HVR-L1) sequence of SEQ ID NO:14 and SEQ ID NO:14 Heavy chain HVR2 (HVR-H2) sequence of ID NO:12. This variant is referred to as the "SIESD" variant (AFD.v8) in the Examples herein below (see, eg, Table 1). In some embodiments, the "SIESD" variant (AFD.v8) comprises the light chain sequence of SEQ ID NO:26 and the heavy chain sequence of SEQ ID NO:27. In some embodiments, the Cys modified version of the "SIESD" variant comprises the light chain sequence of SEQ ID NO:26 and the heavy chain sequence of SEQ ID NO:30. In some embodiments, the "SIESD" variant of the Cys-Pro-Pro-Cys modified version comprises the light chain sequence of SEQ ID NO:26 and the heavy chain sequence of SEQ ID NO:31. In some embodiments, a modified version of a "SIESD" variant comprises a light chain sequence of SEQ ID NO:26 and a heavy chain sequence selected from the group consisting of SEQ ID NOs:75-92 and 117.
在一些方面，用于本发明的缀合物的抗-因子D抗体变体是参比抗-因子D抗体的变体，其中所述参比抗-因子D抗体包含SEQ ID NO:1的轻链序列和选自由以下组成的组的重链序列：SEQ ID NO:34-53和115，并且其中所述变体相对于参比抗-因子D抗体包含以下序列修饰：SEQ ID NO:14的轻链HVR1(HVR-L1)序列和SEQ ID NO:12的重链HVR2(HVR-H2)序列。此种变体被称为“修饰的SIESD”变体。这些修饰的SIESD变体包含不同于SIESD变体的重链恒定结构域，并且其选自由以下组成的组：SEQ ID NO:55-74和116。In some aspects, the anti-Factor D antibody variant used in the conjugates of the invention is a variant of a reference anti-Factor D antibody, wherein the reference anti-Factor D antibody comprises the light of SEQ ID NO: 1 chain sequence and a heavy chain sequence selected from the group consisting of: SEQ ID NO:34-53 and 115, and wherein the variant comprises the following sequence modification relative to the reference anti-factor D antibody: SEQ ID NO:14 Light chain HVR1 (HVR-L1 ) sequence and heavy chain HVR2 (HVR-H2) sequence of SEQ ID NO:12. Such variants are referred to as "modified SIESD" variants. These modified SIESD variants comprise a heavy chain constant domain different from the SIESD variants and are selected from the group consisting of: SEQ ID NOS:55-74 and 116.
在一些方面，用于本发明的缀合物的抗-因子D抗体变体是参比抗-因子D抗体的变体，其中所述参比抗-因子D抗体包含SEQ ID NO:1的轻链序列和SEQ ID NO:2的重链序列，并且其中所述变体相对于参比抗-因子D抗体包含以下序列修饰：SEQ ID NO:14的轻链HVR1(HVR-L1)序列、SEQ ID NO:12的重链HVR2(HVR-H2)序列和SEQ ID NO:15的重链HVR3(HVR-H3)序列。此种变体在本文以下的实施例中被称为“SIESD.N103S”变体(AFD.v14)(参见，例如，表1)。在一些实施方案中，“SIESD.N103S”变体(AFD.v14)包含SEQ ID NO:28的轻链序列和SEQ ID NO:29的重链序列。在一些实施方案中，Cys修饰版本的“SIESD.N103S”变体包含SEQ ID NO:28的轻链序列和SEQ ID NO:32的重链序列。在一些实施方案中，Cys-Pro-Pro-Cys修饰版本的“SIESD.N103S”变体包含SEQ ID NO:28的轻链序列和SEQ ID NO:33的重链序列。在一些实施方案中，修饰版本的“SIESD.N103S”变体包含SEQ ID NO:28的轻链序列和选自由以下组成的组的重链序列：SEQ ID NO:93-110和118。In some aspects, the anti-Factor D antibody variant used in the conjugates of the invention is a variant of a reference anti-Factor D antibody, wherein the reference anti-Factor D antibody comprises the light of SEQ ID NO: 1 chain sequence and the heavy chain sequence of SEQ ID NO:2, and wherein the variant comprises the following sequence modifications relative to the reference anti-Factor D antibody: light chain HVR1 (HVR-L1) sequence of SEQ ID NO:14, SEQ ID NO:14 The heavy chain HVR2 (HVR-H2) sequence of ID NO: 12 and the heavy chain HVR3 (HVR-H3) sequence of SEQ ID NO: 15. This variant is referred to as the "SIESD.N103S" variant (AFD.v14) in the Examples herein below (see, eg, Table 1). In some embodiments, the "SIESD.N103S" variant (AFD.v14) comprises the light chain sequence of SEQ ID NO:28 and the heavy chain sequence of SEQ ID NO:29. In some embodiments, the Cys modified version of the "SIESD.N103S" variant comprises the light chain sequence of SEQ ID NO:28 and the heavy chain sequence of SEQ ID NO:32. In some embodiments, the "SIESD.N103S" variant of the Cys-Pro-Pro-Cys modified version comprises the light chain sequence of SEQ ID NO:28 and the heavy chain sequence of SEQ ID NO:33. In some embodiments, the modified version of the "SIESD.N103S" variant comprises the light chain sequence of SEQ ID NO:28 and the heavy chain sequence selected from the group consisting of SEQ ID NOs:93-110 and 118.
在一些方面，用于本发明的缀合物的抗-因子D抗体变体是参比抗-因子D抗体的变体，其中所述参比抗-因子D抗体包含SEQ ID NO:1的轻链序列和选自由以下组成的组的重链序列：SEQ ID NO:34-53和115，并且其中所述变体相对于参比抗-因子D抗体包含以下序列修饰：SEQ ID NO:14的轻链HVR1(HVR-L1)序列、SEQ ID NO:12的重链HVR2(HVR-H2)序列和SEQ ID NO:15的重链HVR3(HVR-H3)序列。此种变体被称为“修饰的SIESD.N103S”变体。这些修饰的SIESD.N103S变体包含不同于SIESD.N103S变体的重链恒定结构域，并且其选自由以下组成的组：SEQ ID NO:55-74和116。In some aspects, the anti-Factor D antibody variant used in the conjugates of the invention is a variant of a reference anti-Factor D antibody, wherein the reference anti-Factor D antibody comprises the light of SEQ ID NO: 1 chain sequence and a heavy chain sequence selected from the group consisting of: SEQ ID NO:34-53 and 115, and wherein the variant comprises the following sequence modification relative to the reference anti-factor D antibody: SEQ ID NO:14 Light chain HVR1 (HVR-L1 ) sequence, heavy chain HVR2 (HVR-H2) sequence of SEQ ID NO:12, and heavy chain HVR3 (HVR-H3) sequence of SEQ ID NO:15. This variant is referred to as the "modified SIESD.N103S" variant. These modified SIESD.N103S variants comprise a heavy chain constant domain that differs from the SIESD.N103S variant and is selected from the group consisting of SEQ ID NOs: 55-74 and 116.
在一些方面，本发明涉及包含一个或多个抗-因子D抗体变体的缀合物，所述抗-因子D抗体变体包含在参比抗-因子D抗体的HVR内的一个或多个置换。在一些方面，参比抗-因子D抗体包含以下HVR序列：In some aspects, the invention relates to conjugates comprising one or more anti-Factor D antibody variants comprising one or more replacement. In some aspects, the reference anti-Factor D antibody comprises the following HVR sequences:
HVR-L1：ITSTDIDDDMN(SEQ ID NO:5)；HVR-L1: ITSTDIDDDMN (SEQ ID NO: 5);
HVR-L2：GGNTLRP(SEQ ID NO:6)；HVR-L2: GGNTLRP (SEQ ID NO: 6);
HVR-L3：LQSDSLPYT(SEQ ID NO:7)；HVR-L3: LQSDSLPYT (SEQ ID NO: 7);
HVR-H1：GYTFTNYGMN(SEQ ID NO:8)；HVR-H1: GYTFTNYGMN (SEQ ID NO: 8);
HVR-H2：WINTYTGETTYADDFKG(SEQ ID NO:9)；和HVR-H2: WINTYTGETTYADDFKG (SEQ ID NO:9); and
HVR-H3：EGGVNN(SEQ ID NO:10)。HVR-H3: EGGVNN (SEQ ID NO: 10).
相应的变体包含一个或多个以下置换：Corresponding variants contain one or more of the following substitutions:
(a)SEQ ID NO:5中的D5S；(a) D5S among SEQ ID NO:5;
(b)SEQ ID NO:5中的D7E；(b) D7E among SEQ ID NO:5;
(c)SEQ ID NO:5中的D8S(SEQ ID NO:22中公开的a、b和c)；(c) D8S in SEQ ID NO:5 (a, b and c disclosed in SEQ ID NO:22);
(d)SEQ ID NO:9中的D13E(SEQ ID NO:23)；(d) D13E in SEQ ID NO:9 (SEQ ID NO:23);
(e)SEQ ID NO:7中的D4E(SEQ ID NO:24)；或(e) D4E in SEQ ID NO:7 (SEQ ID NO:24); or
(f)SEQ ID NO:10中的N5S(SEQ ID NO:25)。(f) N5S in SEQ ID NO: 10 (SEQ ID NO: 25).
在一些实施方案中，参比抗-因子D抗体包含选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:54-74和116。In some embodiments, the reference anti-Factor D antibody comprises a heavy chain constant domain amino acid sequence selected from the group consisting of SEQ ID NOS:54-74 and 116.
在一些方面，变体结合以上置换(b)-(d)。在另一个方面，变体结合以上置换(b)-(e)。在另一个方面，变体结合以上置换(a)-(d)。在另一个方面，变体结合以上置换(a)-(d)和(f)。在另一个方面，变体包含以上置换(a),(b),(c),(d),(e),or(f)中的一个或多个，并且进一步包含选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:54-74和116。在另一个方面，变体包含选自由以下组成的组的置换：以上置换(b)-(d)、以上置换(b)-(e)、以上置换(a)-(d)以及以上置换(a)-(d)和(f)，其中所述变体进一步包含选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:54-74和116。In some aspects, the variant combines substitutions (b)-(d) above. In another aspect, the variant incorporates substitutions (b)-(e) above. In another aspect, the variant incorporates substitutions (a)-(d) above. In another aspect, the variant combines substitutions (a)-(d) and (f) above. In another aspect, the variant comprises one or more of the above substitutions (a), (b), (c), (d), (e), or (f), and further comprises a group selected from the group consisting of Amino acid sequences of heavy chain constant domains: SEQ ID NOs: 54-74 and 116. In another aspect, the variant comprises a substitution selected from the group consisting of: the above substitutions (b)-(d), the above substitutions (b)-(e), the above substitutions (a)-(d), and the above substitutions ( a)-(d) and (f), wherein said variant further comprises a heavy chain constant domain amino acid sequence selected from the group consisting of SEQ ID NO:54-74 and 116.
在一些方面，本发明涉及包含一个或多个抗-因子D抗体的缀合物，所述抗-因子D抗体包含SEQ ID NO:16、18或19的轻链可变结构域氨基酸序列。在另一个方面，本发明涉及包含抗-因子D抗体的缀合物，所述抗-因子D抗体包含SEQ ID NO:17或20的重链可变结构域氨基酸序列。在另一个方面，抗-因子D抗体可以包含选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:54-74和116。在另一个方面，抗-因子D抗体包含SEQ ID NO:16、18或19的轻链可变结构域氨基酸序列和SEQ ID NO:17或20的重链可变结构域氨基酸序列。例如，抗-因子D抗体可以是包含SEQ ID NO:16的轻链可变结构域氨基酸序列和SEQ ID NO:17的重链可变结构域氨基酸序列的“TM”变体(AFD.v6)；包含SEQ ID NO:18的轻链可变结构域氨基酸序列和SEQ ID NO:17的重链可变结构域氨基酸序列的“TM.D92E”变体(AFD.v7)；包含SEQ ID NO:19的轻链可变结构域氨基酸序列和SEQ ID NO:17的重链可变结构域氨基酸序列的“SIESD”变体(AFD.v8)；或包含SEQ ID NO:19的轻链可变结构域氨基酸序列和SEQID NO:20的重链可变结构域氨基酸序列的“SIESD.N103S”变体(AFD.v14)。In some aspects, the invention relates to conjugates comprising one or more anti-Factor D antibodies comprising the light chain variable domain amino acid sequence of SEQ ID NO: 16, 18 or 19. In another aspect, the invention relates to a conjugate comprising an anti-Factor D antibody comprising the heavy chain variable domain amino acid sequence of SEQ ID NO: 17 or 20. In another aspect, an anti-Factor D antibody can comprise a heavy chain constant domain amino acid sequence selected from the group consisting of SEQ ID NOS:54-74 and 116. In another aspect, the anti-Factor D antibody comprises the light chain variable domain amino acid sequence of SEQ ID NO: 16, 18 or 19 and the heavy chain variable domain amino acid sequence of SEQ ID NO: 17 or 20. For example, the anti-Factor D antibody can be a "TM" variant (AFD.v6) comprising the light chain variable domain amino acid sequence of SEQ ID NO: 16 and the heavy chain variable domain amino acid sequence of SEQ ID NO: 17 ; comprising the light chain variable domain amino acid sequence of SEQ ID NO: 18 and the "TM.D92E" variant (AFD.v7) of the heavy chain variable domain amino acid sequence of SEQ ID NO: 17; comprising SEQ ID NO: A "SIESD" variant (AFD.v8) of the light chain variable domain amino acid sequence of 19 and the heavy chain variable domain amino acid sequence of SEQ ID NO: 17; or a light chain variable structure comprising SEQ ID NO: 19 Domain amino acid sequence and the "SIESD.N103S" variant (AFD.v14) of the heavy chain variable domain amino acid sequence of SEQ ID NO:20.
在另一个方面，抗-因子D抗体包含SEQ ID NO:16、18或19的轻链可变结构域氨基酸序列、SEQ ID NO:17或20的重链可变结构域氨基酸序列和选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:54-74和116。例如，抗-因子D抗体可以是修饰版本的“TM”变体(AFD.v6)，其包含SEQ ID NO:16的轻链可变结构域氨基酸序列、SEQ ID NO:17的重链可变结构域氨基酸序列和选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:55-74和116；修饰版本的“TM.D92E”变体(AFD.v7)，其包含SEQ ID NO:18的轻链可变结构域氨基酸序列、SEQ ID NO:17的重链可变结构域氨基酸序列和选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:55-74和116；修饰版本的“SIESD”变体(AFD.v8)，其包含SEQ ID NO:19的轻链可变结构域氨基酸序列、SEQ ID NO:17的重链可变结构域氨基酸序列和选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:55-74和116；修饰版本的“SIESD.N103S”变体(AFD.v14)，其包含SEQ ID NO:19的轻链可变结构域氨基酸序列、SEQID NO:20的重链可变结构域氨基酸序列和选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:55-74和116。In another aspect, the anti-Factor D antibody comprises the light chain variable domain amino acid sequence of SEQ ID NO: 16, 18 or 19, the heavy chain variable domain amino acid sequence of SEQ ID NO: 17 or 20 and is selected from the group consisting of The heavy chain constant domain amino acid sequences of the group consisting of: SEQ ID NO:54-74 and 116. For example, the anti-Factor D antibody can be a modified version of the "TM" variant (AFD.v6) comprising the light chain variable domain amino acid sequence of SEQ ID NO: 16, the heavy chain variable domain of SEQ ID NO: 17 Domain amino acid sequence and heavy chain constant domain amino acid sequence selected from the group consisting of: SEQ ID NOs: 55-74 and 116; a modified version of the "TM.D92E" variant (AFD.v7) comprising SEQ ID The light chain variable domain amino acid sequence of NO: 18, the heavy chain variable domain amino acid sequence of SEQ ID NO: 17 and the heavy chain constant domain amino acid sequence selected from the group consisting of: SEQ ID NO: 55-74 and 116; a modified version of the "SIESD" variant (AFD.v8) comprising the light chain variable domain amino acid sequence of SEQ ID NO: 19, the heavy chain variable domain amino acid sequence of SEQ ID NO: 17, and selected Heavy chain constant domain amino acid sequence from the group consisting of: SEQ ID NO:55-74 and 116; a modified version of the "SIESD.N103S" variant (AFD.v14) comprising the light chain of SEQ ID NO:19 A variable domain amino acid sequence, a heavy chain variable domain amino acid sequence of SEQ ID NO: 20, and a heavy chain constant domain amino acid sequence selected from the group consisting of SEQ ID NOs: 55-74 and 116.
在一些实施方案中，抗-因子D抗体是修饰版本的“SIESD”变体(AFD.v8)，其包含SEQ ID NO:19的轻链可变结构域氨基酸序列和选自由以下组成的组的重链序列：SEQ IDNO:30、31、75-92和117。在一些实施方案中，抗-因子D抗体是修饰版本的“SIESD”变体(AFD.v8)，其包含SEQ ID NO:26的轻链序列和具有选自由以下组成的组的氨基酸序列的重链：SEQ ID NO:30、31、75-92和117。在另一个实施方案中，抗-因子D抗体是修饰版本的“SIESD.N103S”变体(AFD.v14)，其包含SEQ ID NO:19的轻链可变结构域氨基酸序列和具有选自由以下组成的组的氨基酸序列的重链序列：SEQ ID NO:32、33、93-110和118。在一些实施方案中，抗-因子D抗体是修饰版本的“SIESD.N103S”变体(AFD.v14)，其包含SEQ ID NO:28的轻链序列和选自由以下组成的组的重链序列：SEQ ID NO:32、33、93-110和118。In some embodiments, the anti-Factor D antibody is a modified version of the "SIESD" variant (AFD.v8) comprising the light chain variable domain amino acid sequence of SEQ ID NO: 19 and an Heavy chain sequences: SEQ ID NO: 30, 31, 75-92 and 117. In some embodiments, the anti-Factor D antibody is a modified version of a "SIESD" variant (AFD.v8) comprising a light chain sequence of SEQ ID NO: 26 and a heavy chain having an amino acid sequence selected from the group consisting of Chains: SEQ ID NO:30, 31, 75-92 and 117. In another embodiment, the anti-Factor D antibody is a modified version of the "SIESD.N103S" variant (AFD.v14) comprising the light chain variable domain amino acid sequence of SEQ ID NO: 19 and having an amino acid sequence selected from the group consisting of The heavy chain sequence of the amino acid sequence consisting of the group: SEQ ID NO: 32, 33, 93-110 and 118. In some embodiments, the anti-Factor D antibody is a modified version of the "SIESD.N103S" variant (AFD.v14) comprising a light chain sequence of SEQ ID NO: 28 and a heavy chain sequence selected from the group consisting of : SEQ ID NO: 32, 33, 93-110 and 118.
在一些方面，本发明涉及包含一个或多个抗-因子D抗体的缀合物，所述抗-因子D抗体具有可变轻链和可变重链，所述可变轻链包含具有SEQ ID NO:11或14的序列的HVR-L1，具有SEQ ID NO:6的序列的HVR-L2，和具有SEQ ID NO:7或13的序列的HVR-L3；所述可变重链包含具有SEQ ID NO:8的序列的HVR-H1，具有SEQ ID NO:9或12的序列的HVR-H2，和具有SEQ ID NO:10或15的序列的HVR-H3。在另一个实施方案中，抗-因子D抗体还可以包含选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:54-74和116。例如，抗-因子D抗体可以是包含以下六个HVR序列的“SIESD”变体(AFD.v8)：HVR-L1(SEQ ID NO:14)，HVR-L2(SEQ ID NO:6)，HVR-L3(SEQ ID NO:7)，HVR-H1(SEQ ID NO:8)，HVR-H2(SEQ ID NO:12)，和HVR-H3(SEQ ID NO:10)；或包含以下六个HVR序列的“SIESD.N103S”变体(AFD.v14)：HVR-L1(SEQ ID NO:14)，HVR-L2(SEQ ID NO:6)，HVR-L3(SEQ ID NO:7)，HVR-H1(SEQ ID NO:8)，HVR-H2(SEQ ID NO:12)，和HVR-H3(SEQ ID NO:15)。在一些实施方案中，抗-因子D抗体可以是包含以下六个HVR序列的修饰版本的“SIESD”变体(AFD.v8)：HVR-L1(SEQ ID NO:14)，HVR-L2(SEQ ID NO:6)，HVR-L3(SEQ ID NO:7)，HVR-H1(SEQ ID NO:8)，HVR-H2(SEQ ID NO:12)，和HVR-H3(SEQ ID NO:10)，并且其进一步包含选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:55-74和116。在另一个实施方案中，抗-因子D抗体可以是包含以下六个HVR序列的修饰版本的“SIESD.N103S”变体(AFD.v14)：HVR-L1(SEQ ID NO:14)，HVR-L2(SEQ ID NO:6)，HVR-L3(SEQ ID NO:7)，HVR-H1(SEQ ID NO:8)，HVR-H2(SEQ ID NO:12)，和HVR-H3(SEQ ID NO:15)，并且其进一步包含选自由以下组成的组的重链恒定结构域氨基酸序列：SEQ ID NO:55-74和116。In some aspects, the invention relates to conjugates comprising one or more anti-Factor D antibodies having a variable light chain and a variable heavy chain, the variable light chain comprising The HVR-L1 of the sequence of NO:11 or 14, the HVR-L2 with the sequence of SEQ ID NO:6, and the HVR-L3 with the sequence of SEQ ID NO:7 or 13; HVR-H1 having the sequence of ID NO:8, HVR-H2 having the sequence of SEQ ID NO:9 or 12, and HVR-H3 having the sequence of SEQ ID NO:10 or 15. In another embodiment, the anti-Factor D antibody may further comprise a heavy chain constant domain amino acid sequence selected from the group consisting of SEQ ID NOS:54-74 and 116. For example, an anti-Factor D antibody can be a "SIESD" variant (AFD.v8) comprising the following six HVR sequences: HVR-L1 (SEQ ID NO: 14), HVR-L2 (SEQ ID NO: 6), HVR -L3 (SEQ ID NO:7), HVR-H1 (SEQ ID NO:8), HVR-H2 (SEQ ID NO:12), and HVR-H3 (SEQ ID NO:10); or comprising the following six HVRs "SIESD.N103S" variant (AFD.v14) of the sequence: HVR-L1 (SEQ ID NO: 14), HVR-L2 (SEQ ID NO: 6), HVR-L3 (SEQ ID NO: 7), HVR- H1 (SEQ ID NO:8), HVR-H2 (SEQ ID NO:12), and HVR-H3 (SEQ ID NO:15). In some embodiments, the anti-Factor D antibody may be a "SIESD" variant (AFD.v8) comprising a modified version of the following six HVR sequences: HVR-L1 (SEQ ID NO: 14), HVR-L2 (SEQ ID NO: 14), HVR-L2 (SEQ ID NO:6), HVR-L3 (SEQ ID NO:7), HVR-H1 (SEQ ID NO:8), HVR-H2 (SEQ ID NO:12), and HVR-H3 (SEQ ID NO:10) , and it further comprises a heavy chain constant domain amino acid sequence selected from the group consisting of: SEQ ID NO:55-74 and 116. In another embodiment, the anti-Factor D antibody may be a modified version of the "SIESD.N103S" variant (AFD.v14) comprising the following six HVR sequences: HVR-L1 (SEQ ID NO: 14), HVR- L2 (SEQ ID NO:6), HVR-L3 (SEQ ID NO:7), HVR-H1 (SEQ ID NO:8), HVR-H2 (SEQ ID NO:12), and HVR-H3 (SEQ ID NO : 15), and it further comprises a heavy chain constant domain amino acid sequence selected from the group consisting of: SEQ ID NO: 55-74 and 116.
在一些方面，本发明涉及包含不具有可检测的Asp异构化的抗-因子D抗体变体的缀合物，其中所述变体通过用于除去或减少异构化的方法制备，所述方法包括：(a)鉴定在参比抗-因子D抗体的HVR内的一个或多个倾向于Asp异构化的Asp残基；(b)用Glu置换步骤(a)中鉴定的Asp残基；(c)针对Asp异构化筛选所得的候选变体；和(d)选择不具有可检测的Asp异构化的那些变体。在一些方面，将以上方法与用于除去或减少脱酰胺的方法相结合，所述方法包括(a)鉴定在参比抗-因子D抗体的HVR内的一个或多个倾向于脱酰胺的Asn残基；(b)用Ser置换步骤(a)中鉴定的Asn残基；(c)针对脱酰胺筛选所得的候选变体；和(d)选择具有减少的或消除的脱酰胺的那些变体。在另一个方面，将用于除去或减少异构化的方法与用于减少抗体总体电荷的方法相结合，所述方法是通过：(a)选择在参比抗-因子D抗体的HVR内的一个或多个带负电的氨基酸残基D或E；(b)用Ser置换步骤(a)中选择的残基；(c)针对溶解性筛选所得的候选变体；和(d)选择与所述参比抗-因子D抗体相比具有改善的溶解性的那些变体。In some aspects, the invention relates to conjugates comprising an anti-Factor D antibody variant that does not have detectable Asp isomerization, wherein said variant is prepared by a method for removing or reducing isomerization, said The method comprises: (a) identifying one or more Asp residues within the HVR of a reference anti-Factor D antibody that are prone to Asp isomerization; (b) replacing the Asp residues identified in step (a) with Glu ; (c) screening the resulting candidate variants for Asp isomerization; and (d) selecting those variants that do not have detectable Asp isomerization. In some aspects, the above methods are combined with methods for removing or reducing deamidation comprising (a) identifying one or more Asn(s) within the HVR of a reference anti-Factor D antibody that is prone to deamidation (b) replace the Asn residue identified in step (a) with Ser; (c) screen the resulting candidate variants for deamidation; and (d) select those variants with reduced or eliminated deamidation . In another aspect, the method for removing or reducing isomerization is combined with the method for reducing the overall charge of the antibody by: (a) selecting the isomerization within the HVR of the reference anti-Factor D antibody one or more negatively charged amino acid residues D or E; (b) replacing the residue selected in step (a) with Ser; (c) screening the resulting candidate variant for solubility; Those variants that have improved solubility compared to the reference anti-Factor D antibody.
在一些方面，本发明涉及包含本文公开的一种或多种抗-因子D抗体或抗体变体和一种或多种多臂多元醇的缀合物，其中所述缀合物通过将至少一个本文中公开的抗-因子D抗体或抗体变体共价连接至多元醇来制备。在一些实施方案中，多臂多元醇是PEG。在一些实施方案中，PEG是八聚体。在一些实施方案中，PEG具有通式(Ia)、(Ib)、(IIa)、(IIIa)或(IVa)的结构(如本文中所示)：In some aspects, the invention relates to conjugates comprising one or more anti-Factor D antibodies or antibody variants disclosed herein and one or more multi-armed polyols, wherein the conjugates are obtained by combining at least one The anti-Factor D antibodies or antibody variants disclosed herein are prepared by covalently linking to polyols. In some embodiments, the multi-armed polyol is PEG. In some embodiments, PEG is an octamer. In some embodiments, PEG has the structure (as shown herein) of general formula (Ia), (Ib), (IIa), (Ilia) or (IVa):
在一些方面，当与所述参比抗-因子D抗体相比时，本发明的缀合物包含在保持因子D结合亲和力的同时具有改善的稳定性的抗-因子D抗体变体。在一些方面，抗体以至少约10-9至10-12M的结合亲和力结合因子D。在一些方面，用于本发明的缀合物的抗体包括人抗体、人源化抗体或嵌合抗体。In some aspects, the conjugates of the invention comprise anti-Factor D antibody variants that have improved stability while maintaining Factor D binding affinity when compared to the reference anti-Factor D antibody. In some aspects, the antibody binds Factor D with a binding affinity of at least about 10−9 to 10−12 M. In some aspects, antibodies for use in the conjugates of the invention include human antibodies, humanized antibodies, or chimeric antibodies.
在一些方面，用于本发明的缀合物的抗体是抗体片段(例如抗原结合片段)。本发明的抗体片段可以是例如Fab、Fab’、F(ab’)2、scFv、(scFv)2、dAb、互补决定区(CDR)片段、线性抗体、单链抗体分子、微抗体、双抗体、或由抗体片段形成的多特异性抗体。In some aspects, the antibodies used in the conjugates of the invention are antibody fragments (eg, antigen-binding fragments). Antibody fragments of the invention may be, for example, Fab, Fab', F(ab')2 , scFv, (scFv)2 , dAb, complementarity determining region (CDR) fragments, linear antibodies, single chain antibody molecules, minibodies, diabodies , or multispecific antibodies formed from antibody fragments.
在本发明的其他方面，本发明包括组合物，所述组合物包含本发明的缀合物。在另一个方面，本发明涉及物质的组合物，所述物质的组合物包含与载体结合的如本文所述的本发明的缀合物。任选地，所述载体是药用载体。In other aspects of the invention, the invention includes compositions comprising a conjugate of the invention. In another aspect, the invention relates to a composition of matter comprising a conjugate of the invention as described herein in association with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
在一些方面，本发明包括药物制剂，所述药物制剂包含治疗有效浓度的本文所述的缀合物。在一些方面，所述药物制剂包含至少约100mg/mL、约100至约150mg/mL、约100至约200mg/mL、约100至约300mg/mL、约100至约400mg/mL、约100至约500mg/ml；至少约200mg/mL、至少约300mg/mL、至少约400mg/mL或至少约500mg/mL的浓度的抗体或抗体变体。在一些方面，制剂中抗体或抗体变体的浓度为约200、250、300、350、400、450或500mg/mL。在一些方面，制剂中抗体或抗体变体的浓度小于约450mg/mL。In some aspects, the invention includes pharmaceutical formulations comprising a therapeutically effective concentration of a conjugate described herein. In some aspects, the pharmaceutical formulation comprises at least about 100 mg/mL, about 100 to about 150 mg/mL, about 100 to about 200 mg/mL, about 100 to about 300 mg/mL, about 100 to about 400 mg/mL, about 100 to about The antibody or antibody variant at a concentration of about 500 mg/ml; at least about 200 mg/mL, at least about 300 mg/mL, at least about 400 mg/mL, or at least about 500 mg/mL. In some aspects, the concentration of the antibody or antibody variant in the formulation is about 200, 250, 300, 350, 400, 450, or 500 mg/mL. In some aspects, the concentration of antibody or antibody variant in the formulation is less than about 450 mg/mL.
本发明的另一个方面是本发明的缀合物或药物制剂用于治疗与过度或不受控的补体激活相关的病症的用途。在一个实施方案中，本发明涉及治疗受试者中的补体相关性病症的方法，所述方法包括向所述受试者施用本发明的缀合物或药物制剂。所述病症包括心肺分流术期间的补体激活；由急性心肌梗死、动脉瘤、卒中、出血性休克、挤压伤、多器官衰竭、低血容量性休克、肠缺血或其它引起缺血的事件之后的缺血-再灌注引起的补体激活。补体激活还已经被证明与炎症病症相关，所述炎症病症诸如严重烧伤、内毒素血症、脓毒性休克、成人呼吸窘迫综合征、血液透析；过敏性休克、严重哮喘、血管性水肿、克罗恩病(Crohn’s disease)、镰状细胞性贫血、链球菌感染后肾小球肾炎和胰腺炎。所述病况可以是不良药物反应、药物变态反应、IL-2诱导的血管渗漏综合征或放射摄影造影剂变态反应的结果。在一个实施方案中，补体相关性病症是系统性的。其还可以包括自身免疫疾病，诸如系统性红斑狼疮、重症肌无力、类风湿性关节炎、阿尔茨海默病(Alzheimer’s disease)和多发性硬化。在另一个实施方案中，补体激活还与移植排斥相关。在另一个实施方案中，补体激活还与眼病(病理学涉及补体(包括补体的经典和旁路途径)的所有眼部病况和疾病)或补体相关性眼部病况相关，诸如，例如，不限于，黄斑变性病，诸如所有阶段的年龄相关性黄斑变性(AMD)，包括干性和湿性(非渗出性和渗出性)形式，糖尿病性视网膜病和其它缺血相关性视网膜病，脉络膜新血管形成(CNV)，葡萄膜炎，糖尿病性黄斑水肿，病理性近视，希佩尔-林道病(von Hippel-Lindau disease)，眼的组织胞浆菌病，视网膜中央静脉阻塞(CRVO)，角膜新血管形成和视网膜新血管形成。在一个实例中，补体相关性眼部病况包括年龄相关性黄斑变性(AMD)，包括非渗出性(例如中期干性AMD或地图状萎缩(GA))和渗出性(例如湿性AMD(脉络膜新血管形成(CNV))AMD，糖尿病性视网膜病(DR)，眼内炎和葡萄膜炎。在另外的实例中，非渗出性AMD可以包括硬玻璃疣、软玻璃疣、地图状萎缩和/或色素凝集的存在。在另一个实例中，补体相关性眼部病况包括年龄相关性黄斑变性(AMD)，包括早期AMD(例如包括多个小的至一个或多个非渗出性的中等大小的玻璃疣)、中期AMD(例如包括大量的中等玻璃疣至一个或多个大玻璃疣)和晚期AMD(例如包括地图状萎缩或晚期湿性AMD(CNV)。在另外的实例中，中期干性AMD可以包括大的汇合的玻璃疣。在另外的实例中，地图状萎缩可以包括光感受器和/或视网膜色素上皮(RPE)损失。在另外的实例中，地图状萎缩的面积可以是小的或大的和/或可以是在黄斑区域中或在外围视网膜中。在一个实例中，补体相关性眼部病况是中期干性AMD。在一个实例中，补体相关性眼部病况是地图状萎缩。在一个实例中，补体相关性眼部病况是湿性AMD(脉络膜新血管形成(CNV))。在一个实施方案中，使用可植入端口递送系统施用缀合物或药物制剂。在一个实施方案中，通过玻璃体内施用来施用缀合物或药物制剂。在一个实施方案中，所述方法或用途还包括向受试者施用另外的治疗剂，诸如HTRA1拮抗剂，ANG2拮抗剂，TIE2拮抗剂，VEGF拮抗剂，或C1、C2、C3、C4、C5、C6、C7、C8或C9补体组分中一个或多个的拮抗剂。Another aspect of the invention is the use of a conjugate or pharmaceutical formulation of the invention for the treatment of a disorder associated with excessive or uncontrolled complement activation. In one embodiment, the invention relates to a method of treating a complement-associated disorder in a subject, said method comprising administering to said subject a conjugate or pharmaceutical formulation of the invention. Such conditions include complement activation during cardiopulmonary bypass; caused by acute myocardial infarction, aneurysm, stroke, hemorrhagic shock, crush injury, multiorgan failure, hypovolemic shock, intestinal ischemia, or other ischemic events Complement activation following ischemia-reperfusion. Complement activation has also been shown to be associated with inflammatory conditions such as severe burns, endotoxemia, septic shock, adult respiratory distress syndrome, hemodialysis; anaphylactic shock, severe asthma, angioedema, Crohn's Crohn's disease, sickle cell anemia, poststreptococcal glomerulonephritis, and pancreatitis. The condition may be the result of an adverse drug reaction, drug allergy, IL-2-induced vascular leak syndrome, or radiographic contrast media allergy. In one embodiment, the complement-associated disorder is systemic. It can also include autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Alzheimer's disease and multiple sclerosis. In another embodiment, complement activation is also associated with transplant rejection. In another embodiment, complement activation is also associated with ocular diseases (all ocular conditions and diseases whose pathology involves complement, including the classical and alternative pathways of complement) or complement-associated ocular conditions, such as, for example, without limitation , macular degeneration, such as all stages of age-related macular degeneration (AMD), including dry and wet (nonexudative and exudative) forms, diabetic retinopathy and other ischemia-related retinopathy, choroidal neoplasms Angiogenesis (CNV), uveitis, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), cornea Neovascularization and retinal neovascularization. In one example, the complement-associated ocular condition includes age-related macular degeneration (AMD), including non-exudative (eg, intermediate dry AMD or geographic atrophy (GA)) and exudative (eg, wet AMD (choroidal atrophy) Neovascularization (CNV)) AMD, diabetic retinopathy (DR), endophthalmitis, and uveitis. In additional examples, non-exudative AMD can include hard drusen, soft drusen, geographic atrophy, and and/or the presence of pigment agglutination. In another example, the complement-associated ocular condition includes age-related macular degeneration (AMD), including early AMD (e.g., including multiple small to one or more non-exudative moderate large drusen), intermediate AMD (e.g., including numerous medium drusen to one or more large drusen), and advanced AMD (e.g., including geographic atrophy or late wet AMD (CNV). In another example, intermediate dry AMD can include large confluent drusen. In another example, geographic atrophy can include photoreceptor and/or retinal pigment epithelium (RPE) loss. In another example, the area of geographic atrophy can be small or large and/or may be in the macular area or in the peripheral retina. In one example, the complement-associated ocular condition is intermediate dry AMD. In one example, the complement-associated ocular condition is geographic atrophy In one example, the complement-associated ocular condition is wet AMD (choroidal neovascularization (CNV)). In one embodiment, the conjugate or drug formulation is administered using an implantable port delivery system. In one embodiment In, the conjugate or pharmaceutical formulation is administered by intravitreal administration. In one embodiment, the method or use further comprises administering to the subject an additional therapeutic agent, such as an HTRA1 antagonist, an ANG2 antagonist, a TIE2 antagonist , a VEGF antagonist, or an antagonist of one or more of the C1, C2, C3, C4, C5, C6, C7, C8 or C9 complement components.
在另一个方面，本发明提供试剂盒，所述试剂盒包含本发明的缀合物。在一些实施方案中，本发明提供试剂盒，所述试剂盒包含本发明的缀合物及使用说明。在一些实施方案中，本发明涉及试剂盒，所述试剂盒包含本发明的缀合物以及用于施用所述缀合物以治疗补体相关性病症的说明。在一些实施方案中，本发明提供试剂盒，所述试剂盒包含第一容器，所述第一容器包含组合物，所述组合物包含一种或多种本发明的缀合物；和第二容器，所述第二容器包含缓冲剂。在一些实施方案中，所述缓冲剂是药用的。在一些实施方案中，包含本发明的缀合物的组合物还包含载体，在一些实施方案中，所述载体是药用的。在一些实施方案中，所述试剂盒还包含用于将所述组合物(例如，包含一个或多个抗体或其抗体片段(例如抗原结合片段)的缀合物)施用于受试者的说明。在一些实施方案中，所述试剂盒还包含使用所述试剂盒的说明。In another aspect, the invention provides a kit comprising a conjugate of the invention. In some embodiments, the invention provides a kit comprising a conjugate of the invention and instructions for use. In some embodiments, the invention relates to a kit comprising a conjugate of the invention and instructions for administering the conjugate for the treatment of a complement-associated disorder. In some embodiments, the invention provides a kit comprising a first container comprising a composition comprising one or more conjugates of the invention; and a second a container, the second container comprising a buffer. In some embodiments, the buffer is pharmaceutically acceptable. In some embodiments, a composition comprising a conjugate of the invention further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In some embodiments, the kit further comprises instructions for administering the composition (e.g., a conjugate comprising one or more antibodies or antibody fragments thereof (e.g., antigen-binding fragments) thereof) to a subject . In some embodiments, the kit further comprises instructions for using the kit.
在一些方面，本发明涉及制品，所述制品含有可用于治疗、预防和/或诊断补体相关性病症的材料。在一些实施方案中，本发明涉及制品，所述制品包含：(a)容器；(b)容器上的标签；和(c)用所述容器盛放的物质组合物，所述物质组合物包含本发明的缀合物，其中所述容器上的标签指示所述组合物可以用于治疗、预防和/或诊断补体相关性病症。In some aspects, the invention relates to articles of manufacture containing materials useful for the treatment, prevention and/or diagnosis of complement-associated disorders. In some embodiments, the present invention is directed to an article of manufacture comprising: (a) a container; (b) a label on the container; and (c) a composition of matter contained in the container, the composition of matter comprising The conjugate of the present invention, wherein the label on the container indicates that the composition can be used for the treatment, prevention and/or diagnosis of a complement-associated disorder.
在一些方面，本发明提供本发明的缀合物在制备药物中的用途，所述药物用于疾病(诸如补体相关性眼部病况)的治疗性和/或预防性治疗。在一个实施方案中，本发明涉及治疗受试者中的补体相关性病症(诸如补体相关性眼部病况)的方法，所述方法包括向所述受试者施用本发明的缀合物或药物制剂。在一些实施方案中，补体相关性眼部病况选自年龄相关性黄斑变性(AMD)，包括非渗出性(例如中期干性AMD或地图状萎缩(GA))和渗出性(例如湿性AMD(脉络膜新血管形成(CNV))AMD，糖尿病性视网膜病(DR)，眼内炎和葡萄膜炎。在一个实例中，补体相关性眼部病况是中期干性AMD。在一个实例中，补体相关性眼部病况是地图状萎缩。在一个实例中，补体相关性眼部病况是湿性AMD(脉络膜新血管形成(CNV))。In some aspects, the invention provides the use of a conjugate of the invention for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disease, such as a complement-associated ocular condition. In one embodiment, the invention relates to a method of treating a complement-associated disorder, such as a complement-associated ocular condition, in a subject, the method comprising administering to the subject a conjugate or medicament of the invention preparation. In some embodiments, the complement-associated ocular condition is selected from age-related macular degeneration (AMD), including non-exudative (eg, intermediate dry AMD or geographic atrophy (GA)) and exudative (eg, wet AMD) (choroidal neovascularization (CNV)) AMD, diabetic retinopathy (DR), endophthalmitis, and uveitis. In one example, the complement-associated ocular condition is intermediate dry AMD. In one example, the complement The associated ocular condition is geographic atrophy. In one example, the complement-associated ocular condition is wet AMD (choroidal neovascularization (CNV)).
在一些方面，本发明提供本发明的制品在制备药物中的用途，所述药物用于疾病(诸如补体相关性眼部病况)的治疗性和/或预防性治疗。在一些实施方案中，补体相关性眼部病况选自年龄相关性黄斑变性(AMD)，包括非渗出性(例如中期干性AMD或地图状萎缩(GA))和渗出性(例如湿性AMD(脉络膜新血管形成(CNV))AMD，糖尿病性视网膜病(DR)，眼内炎和葡萄膜炎。在一个实例中，补体相关性眼部病况是中期干性AMD。在一个实例中，补体相关性眼部病况是地图状萎缩。在一个实例中，补体相关性眼部病况是湿性AMD(脉络膜新血管形成(CNV))。In some aspects, the invention provides the use of an article of manufacture of the invention for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disease, such as a complement-associated ocular condition. In some embodiments, the complement-associated ocular condition is selected from age-related macular degeneration (AMD), including non-exudative (eg, intermediate dry AMD or geographic atrophy (GA)) and exudative (eg, wet AMD) (choroidal neovascularization (CNV)) AMD, diabetic retinopathy (DR), endophthalmitis, and uveitis. In one example, the complement-associated ocular condition is intermediate dry AMD. In one example, the complement The associated ocular condition is geographic atrophy. In one example, the complement-associated ocular condition is wet AMD (choroidal neovascularization (CNV)).
在一些方面，本发明提供本发明的试剂盒在制备药物中的用途，所述药物用于疾病(诸如补体相关性眼部病况)的治疗性和/或预防性治疗。在一些实施方案中，补体相关性眼部病况选自年龄相关性黄斑变性(AMD)，包括非渗出性(例如中期干性AMD或地图状萎缩(GA))和渗出性(例如湿性AMD(脉络膜新血管形成(CNV))AMD，糖尿病性视网膜病(DR)，眼内炎和葡萄膜炎。在一个实例中，补体相关性眼部病况是中期干性AMD。在一个实例中，补体相关性眼部病况是地图状萎缩。在一个实例中，补体相关性眼部病况是湿性AMD(脉络膜新血管形成(CNV))。In some aspects, the invention provides the use of a kit of the invention for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a disease, such as a complement-associated ocular condition. In some embodiments, the complement-associated ocular condition is selected from age-related macular degeneration (AMD), including non-exudative (eg, intermediate dry AMD or geographic atrophy (GA)) and exudative (eg, wet AMD) (choroidal neovascularization (CNV)) AMD, diabetic retinopathy (DR), endophthalmitis, and uveitis. In one example, the complement-associated ocular condition is intermediate dry AMD. In one example, the complement The associated ocular condition is geographic atrophy. In one example, the complement-associated ocular condition is wet AMD (choroidal neovascularization (CNV)).
在一些方面，本发明提供包含缀合物的制剂，所述缀合物包含一个或多个因子D拮抗剂，并且所述制剂还包含HTRA1拮抗剂，ANG2拮抗剂，TIE2拮抗剂，VEGF拮抗剂，或C1、C2、C3、C4、C5、C6、C7、C8和C9补体组分中一个或多个的拮抗剂。在一些实施方案中，因子D拮抗剂是抗-因子D抗体。在另外的实施方案中，抗-因子D抗体是本文所述的抗-因子D抗体变体。在一些实施方案中，HTRA1拮抗剂是抗-HTRA1抗体。在另一个实施方案中，ANG2拮抗剂是抗-ANG2抗体。在另一个实施方案中，TIE2拮抗剂是抗-TIE2抗体。在另一个实施方案中，VEGF拮抗剂是抗-VEGF抗体。在另一个实施方案中，C2和/或C4和/或C5补体组分的拮抗剂是抗-C2和/或抗-C4和/或抗-C5抗体。In some aspects, the invention provides a formulation comprising a conjugate comprising one or more Factor D antagonists, and the formulation further comprises an HTRA1 antagonist, an ANG2 antagonist, a TIE2 antagonist, a VEGF antagonist , or an antagonist of one or more of the C1, C2, C3, C4, C5, C6, C7, C8 and C9 complement components. In some embodiments, the Factor D antagonist is an anti-Factor D antibody. In additional embodiments, the anti-Factor D antibody is an anti-Factor D antibody variant described herein. In some embodiments, the HTRA1 antagonist is an anti-HTRA1 antibody. In another embodiment, the ANG2 antagonist is an anti-ANG2 antibody. In another embodiment, the TIE2 antagonist is an anti-TIE2 antibody. In another embodiment, the VEGF antagonist is an anti-VEGF antibody. In another embodiment, the antagonist of the C2 and/or C4 and/or C5 complement component is an anti-C2 and/or anti-C4 and/or anti-C5 antibody.
在一些方面，治疗患有与过度或不受控的补体激活相关的病症的人受试者中的与过度或不受控的补体激活相关的病症包括向所述受试者施用有效量的治疗化合物，诸如包含一个或多个因子D拮抗剂的缀合物，并且还包括向所述受试者施用有效量的第二治疗化合物，诸如HTRA1拮抗剂，ANG2拮抗剂，TIE2拮抗剂，VEGF拮抗剂，或C1、C2、C3、C4、C5、C6、C7、C8和C9补体组分中一个或多个的拮抗剂。在一些实施方案中，因子D拮抗剂是抗-因子D抗体。在一些实施方案中，抗-因子D抗体是本文所述的抗-因子D抗体变体。在一些实施方案中，HTRA1拮抗剂是抗-HTRA1抗体。在另一个实施方案中，ANG2拮抗剂是抗-ANG2抗体。在另一个实施方案中，TIE2拮抗剂是抗-TIE2抗体。在另一个实施方案中，VEGF拮抗剂是抗-VEGF抗体。在另一个实施方案中，C2和/或C4和/或C5补体组分的拮抗剂是抗-C2和/或抗-C4和/或抗-C5抗体。In some aspects, treating a disorder associated with excessive or uncontrolled complement activation in a human subject suffering from a disorder associated with excessive or uncontrolled complement activation comprises administering to the subject an effective amount of a therapeutic Compounds, such as conjugates comprising one or more Factor D antagonists, and further comprising administering to said subject an effective amount of a second therapeutic compound, such as an HTRA1 antagonist, an ANG2 antagonist, a TIE2 antagonist, a VEGF antagonist agents, or antagonists of one or more of the C1, C2, C3, C4, C5, C6, C7, C8 and C9 complement components. In some embodiments, the Factor D antagonist is an anti-Factor D antibody. In some embodiments, the anti-Factor D antibody is an anti-Factor D antibody variant described herein. In some embodiments, the HTRA1 antagonist is an anti-HTRA1 antibody. In another embodiment, the ANG2 antagonist is an anti-ANG2 antibody. In another embodiment, the TIE2 antagonist is an anti-TIE2 antibody. In another embodiment, the VEGF antagonist is an anti-VEGF antibody. In another embodiment, the antagonist of the C2 and/or C4 and/or C5 complement component is an anti-C2 and/or anti-C4 and/or anti-C5 antibody.
在一些方面，包含因子D拮抗剂的缀合物和任何第二治疗化合物的施用可以同时进行，例如，作为单个组合物或者作为两种以上的不同组合物，使用相同或不同的施用途径。备选地或另外地，所述施用可以以任何顺序相继进行。In some aspects, the administration of the conjugate comprising the Factor D antagonist and any second therapeutic compound can occur simultaneously, eg, as a single composition or as two or more different compositions, using the same or different routes of administration. Alternatively or additionally, said administrations may be performed sequentially in any order.
附图简述Brief description of the drawings
图1A-1D显示了参比抗-因子D抗体WT(aFD.WT)及其选择变体的氨基酸序列(1A：WT的轻链和重链序列；1B：轻链和重链可变结构域的比对；1C：SIESD(AFD.v8)的轻链和重链序列，以及Cys修饰的SIESD(AFD.v8)和Cys-Pro-Pro-Cys-修饰的SIESD(AFD.v8)的重链序列；1D：SIESD.N103S(AFD.v14)的轻链和重链序列，以及Cys修饰的SIESD.N103S(AFD.v14)和Cys-Pro-Pro-Cys-修饰的SIESD.N103S(AFD.v14))的重链序列)。可变结构域内的HVR是加下划线的。变体中的残基置换以粗体显示。在图1C和1D中，Cys和Cys-Pro-Pro-Cys(SEQ IDNO:21)修饰以斜体显示。Figures 1A-1D show the amino acid sequences of the reference anti-Factor D antibody WT (aFD.WT) and selected variants thereof (1A: light and heavy chain sequences of WT; 1B: light and heavy chain variable domains 1C: Light and heavy chain sequences of SIESD (AFD.v8), and heavy chains of Cys-modified SIESD (AFD.v8) and Cys-Pro-Pro-Cys-modified SIESD (AFD.v8) Sequence; 1D: Light and heavy chain sequences of SIESD.N103S (AFD.v14), and Cys-modified SIESD.N103S (AFD.v14) and Cys-Pro-Pro-Cys-modified SIESD.N103S (AFD.v14 )) of the heavy chain sequence). HVRs within variable domains are underlined. Residue substitutions in variants are shown in bold. In Figures 1C and ID, Cys and Cys-Pro-Pro-Cys (SEQ ID NO: 21 ) modifications are shown in italics.
图2A-AC显示在延长的时间内在限定的条件下不同抗体Fab片段的抗原结合能力(2A：10mg/mL的Fab蛋白浓度，在pH 5.5缓冲剂中；2B：100mg/ml的Fab蛋白浓度，在PBS中；2C：100mg/ml的Fab蛋白浓度，在PBS中)。Figure 2A-AC shows the antigen-binding capacity of different antibody Fab fragments under defined conditions over an extended period of time (2A: Fab protein concentration of 10 mg/mL in pH 5.5 buffer; 2B: Fab protein concentration of 100 mg/ml, in PBS; 2C: Fab protein concentration of 100 mg/ml in PBS).
图3A-3B显示在限定的条件下不同抗体Fab片段随时间的降解，由此通过离子交换色谱法(IEC)确定主峰(3A：10mg/mL的Fab蛋白浓度，在pH 5.5缓冲剂中；3B：100mg/ml的Fab蛋白浓度，在PBS中)。Figures 3A-3B show the degradation of different antibody Fab fragments over time under defined conditions, whereby the main peak was determined by ion exchange chromatography (IEC) (3A: Fab protein concentration of 10 mg/mL in pH 5.5 buffer; 3B : Fab protein concentration of 100 mg/ml in PBS).
图4A-4B显示在限定的条件下不同抗体Fab片段随时间的异构化和脱酰胺(4A：10mg/mL的Fab蛋白浓度，在pH 5.5缓冲剂中；4B：100mg/ml的Fab蛋白浓度，在PBS中)。Figures 4A-4B show isomerization and deamidation of different antibody Fab fragments over time under defined conditions (4A: Fab protein concentration of 10 mg/mL in pH 5.5 buffer; 4B: Fab protein concentration of 100 mg/ml , in PBS).
图5显示在限定的条件下在延长的时间内不同抗体Fab片段的聚集(100mg/ml的Fab蛋白浓度，在PBS中)，如通过分子排阻色谱法(SEC)测量单体峰所确定的。Figure 5 shows the aggregation of different antibody Fab fragments over an extended period of time under defined conditions (Fab protein concentration of 100 mg/ml in PBS), as determined by measuring monomer peaks by size exclusion chromatography (SEC) .
图6显示aFD WT、AFD.v2、AFD.v6和AFD.v8在pH 6以及低离子强度(～100mg/ml，在20mM His-HCl，pH 6中)的溶解性。Figure 6 shows the solubility of aFD WT, AFD.v2, AFD.v6 and AFD.v8 at pH 6 and low ionic strength (-100 mg/ml in 20 mM His-HCl, pH 6).
图7显示抗体Fab片段在pH 6和低离子强度(～100mg/ml，在20mM His-HCl，pH 6中)的溶解性。aFD.WT的不溶性通过经由透析交换到PBS(一种含盐(NaCl)的缓冲剂)中而转变。Figure 7 shows the solubility of antibody Fab fragments at pH 6 and low ionic strength (-100 mg/ml in 20 mM His-HCl, pH 6). Insolubility of aFD.WT was converted by exchange into PBS, a buffer containing saline (NaCl), via dialysis.
图8显示227mg/ml(aFD.WT)，269mg/ml(AFD.v8)和344mg/ml(AFD.v14)的抗体Fab片段在PBS(pH 7.3)中的溶解性。Figure 8 shows the solubility of antibody Fab fragments at 227 mg/ml (aFD.WT), 269 mg/ml (AFD.v8) and 344 mg/ml (AFD.v14) in PBS (pH 7.3).
图9显示在2-8℃温育3周前通过分子排阻色谱法(SEC)测量的SIESD.N103S(AFD.v14)在PBS中的％聚集。Figure 9 shows the % aggregation of SIESD.N103S (AFD.v14) in PBS measured by size exclusion chromatography (SEC) before incubation at 2-8°C for 3 weeks.
图10A显示在37℃在热应力下在延长的时间内高浓度(272mg/mL)AFD.v8制剂(20mM His-HCl，pH 5.5)的抗原结合能力。画有阴影线的面积表示测量中±10％的标准误差。Figure 10A shows the antigen binding capacity of a high concentration (272 mg/mL) AFD.v8 formulation (20 mM His-HCl, pH 5.5) under heat stress at 37°C for an extended period of time. The hatched area represents a standard error of ±10% in the measurement.
图10B显示在37℃在热应力下在延长的时间内高浓度(272mg/mL)AFD.v8制剂(20mM His-HCl，pH 5.5)的化学和物理稳定性。N101和E95根据Kabat编号。Figure 10B shows the chemical and physical stability of a high concentration (272 mg/mL) AFD.v8 formulation (20 mM His-HCl, pH 5.5) under heat stress at 37°C for an extended period of time. N101 and E95 are numbered according to Kabat.
图11显示在兔中进行玻璃体内注射后抗体Fab片段的药物动力学。Figure 11 shows the pharmacokinetics of antibody Fab fragments following intravitreal injection in rabbits.
图12显示在pH 5.5缓冲剂中的抗体Fab片段的粘度的蛋白浓度依赖性。Figure 12 shows the protein concentration dependence of the viscosity of antibody Fab fragments in pH 5.5 buffer.
图13A和13B显示了包含六甘油(HGEO)核心(HGEO-400MA,NOFAmerica,Corp.)和三季戊四醇(TP)核心(8ARM(TP)-PEG-MAL,JenKem Technology,USA)的多臂PEG的MALDI分析(13A：HGEO核心；13B：TP核心)。Figures 13A and 13B show a core containing hexaglycerol (HGEO) ( MALDI analysis of multi-arm PEG of HGEO-400MA, NOFA America, Corp.) and tripentaerythritol (TP) core (8ARM(TP)-PEG-MAL, JenKem Technology, USA) (13A: HGEO core; 13B: TP core).
图14A–14C显示了在20mM His-乙酸盐，pH 5.5，50mM NaCl(等度梯度)在Sephacryl S-300HR(GE Healthcare)柱上通过尺寸排阻色谱法(SEC)纯化AFD.v14.C+TP八聚体的结果(14A：SEC柱的初始色谱图；14B：600mL至1100mL的峰的放大；14C：14B所显示的纯化期间收集的色谱级分的MALS谱)。Figures 14A-14C show purification of AFD.v14.C by size exclusion chromatography (SEC) on a Sephacryl S-300HR (GE Healthcare) column at 20 mM His-acetate, pH 5.5, 50 mM NaCl (isocratic gradient) + TP octamer results (14A: initial chromatogram of the SEC column; 14B: zoom-in of the peak from 600 mL to 1100 mL; 14C: MALS spectrum of the chromatographic fraction collected during purification shown in 14B).
图15A–14C显示了在20mM His-乙酸盐，pH 5.5，50mM NaCl(等度梯度)在Sephacryl S-300HR(GE Healthcare)柱上通过SEC纯化AFD.v14.C+HG八聚体的结果(15A：SEC柱的初始色谱图；15B：2900-3600mL的15A色谱图的放大；15C：图15B所显示的纯化期间收集的色谱级分的MALS谱)。Figures 15A-14C show the results of purification of AFD.v14.C+HG octamer by SEC on a Sephacryl S-300HR (GE Healthcare) column at 20 mM His-acetate, pH 5.5, 50 mM NaCl (isocratic gradient) (15A: initial chromatogram of SEC column; 15B: zoom-in of 15A chromatogram at 2900-3600 mL; 15C: MALS spectrum of chromatographic fractions collected during purification shown in FIG. 15B).
图16A–16B显示了在PBS(pH 7.4)中使用Sephacryl S-400HR(GE Healthcare)柱分析AFD.v14.C+HG八聚体的结果(16A：柱的初始色谱图；16B：图16A色谱图的MALS谱)。Figures 16A–16B show the results of the analysis of AFD.v14.C+HG octamer using a Sephacryl S-400HR (GE Healthcare) column in PBS (pH 7.4) (16A: initial chromatogram of the column; 16B: chromatogram of Figure 16A MALS spectrum of Fig.
图17A–17B显示了在20mM His-乙酸盐，pH 5.5，50mM NaCl中(等度梯度)在Sephacryl S-300HR(GE Healthcare)柱上通过SEC纯化AFD.v14.C+HGEO八聚体的结果，随后是PBS，pH 7.4中以0.25mL/分钟在Sephacryl S-400HR上的SEC-MALS表征(17A：SEC S-400柱的初始色谱图；17B:图17A色谱级分的MALS谱)。Figures 17A-17B show purification of AFD.v14.C+HGEO octamer by SEC on Sephacryl S-300HR (GE Healthcare) column in 20 mM His-acetate, pH 5.5, 50 mM NaCl (isocratic gradient). The results were followed by SEC-MALS characterization on Sephacryl S-400HR at 0.25 mL/min in PBS, pH 7.4 (17A: initial chromatogram of the SEC S-400 column; 17B: MALS profile of the chromatographic fraction of Figure 17A).
图18A–18B显示了在20mM His-乙酸盐，pH 5.5，50mM NaCl中(等度梯度)在Sephacryl S-300HR(GE Healthcare)柱上通过SEC纯化AFD.v14.C+HGEO八聚体的结果，随后是Tosoh G3000PW柱上的SEC-MALS表征(18A：SEC S-300柱的初始色谱图；18B:采用G3000PW柱使用SEC-MALS对18A的S-300级分的激光强度的重叠)。Figures 18A-18B show the purification of AFD.v14.C+HGEO octamer by SEC on Sephacryl S-300HR (GE Healthcare) column in 20mM His-acetate, pH 5.5, 50mM NaCl (isocratic gradient). Results followed by SEC-MALS characterization on Tosoh G3000PW column (18A: initial chromatogram of SEC S-300 column; 18B: overlay of laser intensity of S-300 fraction of 18A using SEC-MALS with G3000PW column).
图19A–19C显示了在50CV内，在Triton洗液、10-20％1M NaCl梯度中，通过阳离子交换色谱法(CEX)进一步纯化实施例9a中的选择级分(图14所示)(19A：CEX柱的初始色谱图；19B：来自CEX柱的级分的SEC凝胶；19C：图19A中色谱级分的MALS谱)。Figures 19A-19C show the further purification of selected fractions in Example 9a (shown in Figure 14) by cation exchange chromatography (CEX) in a Triton wash, 10-20% 1M NaCl gradient within 50CV (19A : initial chromatogram of the CEX column; 19B: SEC gel of the fraction from the CEX column; 19C: MALS spectrum of the chromatographic fraction in Figure 19A).
图20显示了CEX纯化后AFD.v14.C+TP八聚体的最终分析运行。Figure 20 shows the final analytical run of the AFD.v14.C+TP octamer after CEX purification.
图21A–21B比较了阳离子交换色谱(CEX)、SEC S-400HR柱上的SEC色谱或SEC S-300HR柱上的SEC色谱之后，AFD.v14.C+TP八聚体的纯化方法的结果(21A：三种不同纯化柱的色谱的叠加展示；21B：比较来自三种不同纯化柱的样品的SEC凝胶)。Figures 21A–21B compare the results of the AFD.v14.C+TP octamer purification method after cation exchange chromatography (CEX), SEC chromatography on a SEC S-400HR column, or SEC chromatography on a SEC S-300HR column ( 21A: overlay display of chromatograms from three different purification columns; 21B: comparison of SEC gels of samples from three different purification columns).
图22A–22B比较了使用具有不同核心的PEG制备的PEG-Fab缀合物(22A：比较了具有不同核心的缀合物的纯化样品的SEC凝胶；22B：使用不同核心制备的缀合物的MALS谱)。Figures 22A-22B compare PEG-Fab conjugates prepared using PEG with different cores (22A: compares SEC gels of purified samples of conjugates with different cores; 22B: conjugates prepared with different cores MALS spectrum).
图23显示了AFD.v14.C+HG八聚体和由来自NOF America Corp.的PTE-400MA(一种四聚体)制备的PEG-Fab缀合物的粘度作为AFD.v14浓度的函数。Figure 23 shows the AFD.v14.C+HG octamer and the Viscosity of PEG-Fab conjugates prepared from PTE-400MA (a tetramer) as a function of AFD.v14 concentration.
图24显示了在20℃，在20mL His-Ace,pH 6.5和50mM NaCl中，AFD.v14.C+TP八聚体和AFD.v14.C+HGEO八聚体的粘度作为AFD.v14浓度的函数。Figure 24 shows the viscosity of AFD.v14.C+TP octamer and AFD.v14.C+HGEO octamer as a function of AFD.v14 concentration in 20mL His-Ace, pH 6.5 and 50mM NaCl at 20°C. function.
图25A和25B显示了在10mg/mL,PBS(25A)和10mg/mL的20mM组氨酸HCl,50mM NaCl,于pH 6.5(25B)，AFD.v14.C+TP八聚体的热稳定性作为时间的函数。Figures 25A and 25B show the thermal stability of AFD.v14.C+TP octamer at 10 mg/mL, PBS (25A) and 10 mg/mL of 20 mM histidine HCl, 50 mM NaCl, at pH 6.5 (25B) as a function of time.
图26和27显示了Fab片段的缓慢释放和热稳定性研究过程中的二聚化(26：随时间的缀合物的SEC-MALS分析；27：随时间的缀合物的CE-SDS分析)。Figures 26 and 27 show slow release of Fab fragments and dimerization during thermostability studies (26: SEC-MALS analysis of conjugates over time; 27: CE-SDS analysis of conjugates over time ).
图28显示了热稳定性期间AFD.v14.C+TP八聚体与因子D的结合能力的维持，如通过表面等离子共振所测量的。Figure 28 shows the maintenance of the ability of the AFD.v14.C+TP octamer to bind Factor D during thermostability, as measured by surface plasmon resonance.
图29A和29B显示了在药代动力学研究中，在施用AFD.v14或AFD.v14.C+TP八聚体之后，AFD.v14在食蟹猴玻璃体液中的浓度(相对于时间)(29A：玻璃体液浓度；29B：针对给药强度标准化的玻璃体液浓度数据)。Figures 29A and 29B show the concentration of AFD.v14 in the vitreous humor of cynomolgus monkeys (relative to time) after administration of AFD.v14 or AFD.v14.C+TP octamer in a pharmacokinetic study ( 29A: vitreous humor concentration; 29B: vitreous humor concentration data normalized to dosing intensity).
图30A和30B显示了在药代动力学研究中，在施用AFD.v14或AFD.v14.C+TP八聚体之后，AFD.v14在食蟹猴眼睛房水中的浓度(相对于时间)(30A：眼睛房水浓度；30B：针对给药强度标准化的眼睛房水浓度数据)。Figures 30A and 30B show the concentration of AFD.v14 in the aqueous humor of cynomolgus monkey eyes (relative to time) after administration of AFD.v14 or AFD.v14.C+TP octamer in a pharmacokinetic study ( 30A: eye aqueous humor concentration; 30B: eye aqueous humor concentration data normalized to dosing strength).
图31A和31B显示了在药代动力学研究中，在施用AFD.v14或AFD.v14.C+TP八聚体之后，AFD.v14在食蟹猴视网膜匀浆中的浓度(相对于时间)(31A：视网膜浓度；31B：针对给药强度标准化的视网膜浓度数据)。Figures 31A and 31B show the concentration of AFD.v14 in cynomolgus monkey retina homogenates (vs. time) following administration of AFD.v14 or AFD.v14.C+TP octamer in a pharmacokinetic study (31A: retinal concentrations; 31B: retinal concentration data normalized to dose intensity).
图32A–32C显示了在玻璃体内和静脉内注射施用的药代动力学研究中，在施用AFD.v14或AFD.v14.C+TP八聚体之后，AFD.v14在食蟹猴血浆中的浓度(相对于时间)(32A：玻璃体内注射的血浆浓度；32B：针对给药强度标准化的玻璃体内注射血浆浓度；32C：静脉内施用的血浆浓度)。32A-32C show the pharmacokinetic studies of intravitreal and intravenous injection administration, after the administration of AFD.v14 or AFD.v14.C+TP octamer, AFD.v14 in cynomolgus monkey plasma Concentrations (vs. time) (32A: plasma concentrations for intravitreal injection; 32B: plasma concentrations for intravitreal injection normalized to dosing strength; 32C: plasma concentrations for intravenous administration).
图33A–33B显示了通过静脉内或玻璃体内注射施用AFD.v14.C+TP八聚体之后，在药代动力学研究中因子D浓度和AFD.v14.C+TP八聚体的比较(33A：血浆浓度；33B：眼部浓度)。Figures 33A-33B show the comparison of Factor D concentration and AFD.v14.C+TP octamer in pharmacokinetic studies after administration of AFD.v14.C+TP octamer by intravenous or intravitreal injection ( 33A: plasma concentration; 33B: eye concentration).
图34A–34B显示了因子D依赖性因子B激活的时间分辨荧光能量转移(TR-FRET)测量的抑制曲线(34A：Fab-四聚体缀合物，与未缀合的Fab相比；34B：AFD.v14.C+TP八聚体，与未缀合的Fab相比)。Figures 34A-34B show the inhibition curves of factor D-dependent factor B activation time-resolved fluorescence energy transfer (TR-FRET) measurements (34A: Fab-tetramer conjugate, compared to unconjugated Fab; 34B : AFD.v14.C+TP octamer compared to unconjugated Fab).
图35A–35E显示了在玻璃体内注射施用之后，相比于食蟹猴血浆中的总因子D和治疗剂浓度的系统性AP补体活性(35A：10mg/眼lampalizumab(对比数据)；35B：25mg/眼AFD.v14；35C：3.9mg/眼AFD.v14.C+TP八聚体；35D：7.1mg/眼AFD.v14.C+HG八聚体；35E：11.8mg/眼AFD.v14.C+HG八聚体)。Figures 35A-35E show systemic AP complement activity compared to total Factor D and therapeutic agent concentrations in cynomolgus monkey plasma following intravitreal injection administration (35A: 10 mg/eye lampalizumab (comparative data); 35B: 25 mg /eye AFD.v14; 35C: 3.9mg/eye AFD.v14.C+TP octamer; 35D: 7.1mg/eye AFD.v14.C+HG octamer; 35E: 11.8mg/eye AFD.v14. C+HG octamer).
发明详述Detailed description of the invention
定义definition
贯穿本申请使用的术语被解释为具有对本领域技术人员来说普通的和典型的含义。然而，申请人希望的是，以下术语被给予以下限定的具体定义。Terms used throughout this application are to be interpreted to have ordinary and typical meanings for those skilled in the art. However, it is the applicant's desire that the following terms be given the specific definitions defined below.
术语“抗体”以最广涵义使用，并且具体地涵盖全长单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要它们显示所需的生物学活性(诸如抗原结合活性)即可。抗体(Ab)和免疫球蛋白(Ig)是具有相同结构特性的糖蛋白。抗体显示对特定靶标的结合特异性，而免疫球蛋白包括抗体和其它缺少靶标特异性的抗体样分子。天然抗体和免疫球蛋白通常是约150,000道尔顿的异源四聚体糖蛋白，由两条相同的轻(L)链和两条相同的重(H)链组成。每条重链在一端具有可变结构域(VH)，之后是若干个恒定结构域。每条轻链在一端具有可变结构域(VL)而在其另一端具有恒定结构域。如本文所使用的术语“抗体”清楚地涵盖保持抗原结合活性的抗体片段。The term "antibody" is used in the broadest sense and specifically encompasses full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired biological activity ( such as antigen binding activity). Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins that share the same structural properties. Antibodies exhibit binding specificity for a particular target, while immunoglobulins include antibodies and other antibody-like molecules that lack target specificity. Native antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (VH ) at one end followed by several constant domains. Each light chain has a variable domain (VL ) at one end and a constant domain at its other end. The term "antibody" as used herein expressly encompasses fragments of antibodies that retain antigen binding activity.
“抗体片段”是指不同于完整抗体的包含完整抗体的一部分的分子，所述部分结合与完整抗体结合的抗原。抗体片段的实例包括但不限于Fv、Fab、Fab-SH、Fab’-SH、Fab’、Fab-C、Fab’-C、Fab’-C-SH、Fab-C-SH、scFv、双抗体(diabody)或F(ab’)2；双抗体；线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。"Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab-SH, Fab'-SH, Fab', Fab-C, Fab'-C, Fab'-C-SH, Fab-C-SH, scFv, diabody (diabody) or F(ab')2 ; diabodies; linear antibodies; single-chain antibody molecules (eg, scFv); and multispecific antibodies formed from antibody fragments.
如本文所使用的，“抗-因子D抗体”意指以这样的方式特异性结合因子D的抗体，从而抑制或基本上减少补体激活。As used herein, "anti-Factor D antibody" means an antibody that specifically binds Factor D in such a manner as to inhibit or substantially reduce complement activation.
术语“因子D”用在本文中指天然序列和变体因子D多肽。The term "Factor D" is used herein to refer to native sequence and variant Factor D polypeptides.
如本文所使用的，术语“AFD.Ab”是指任何抗-因子D抗体。As used herein, the term "AFD.Ab" refers to any anti-Factor D antibody.
如本文所使用的，“Fab”是指这样的抗体：所述抗体包含重链恒定区，所述重链恒定区包含CH1结构域或CH1结构域的足够的部分以与轻链恒定区形成二硫键，但是不含有CH2结构域或CH3结构域。如本文所使用的，Fab可以包含铰链区的一个或多个氨基酸。因此，如本文所使用的，术语“Fab”涵盖Fab’抗体。Fab可以包含另外的非天然氨基酸，诸如C-末端半胱氨酸，在该情况下其可以称为Fab-C。如下讨论的，术语Fab-C还涵盖包含铰链区的天然氨基酸(包括C-末端处的天然半胱氨酸)的Fab。在一些实施方案中，Fab包含工程改造的半胱氨酸(即，Fab可以是THIOMAB)。As used herein, "Fab" refers to an antibody comprising a heavy chain constant region comprising a CH1 domain or a sufficient portion of a CH1 domain to form a binary region with a light chain constant region. Sulfur bonds, but no CH2 or CH3 domains. As used herein, a Fab may comprise one or more amino acids of the hinge region. Thus, as used herein, the term "Fab" encompasses Fab' antibodies. A Fab may contain additional unnatural amino acids, such as a C-terminal cysteine, in which case it may be referred to as Fab-C. As discussed below, the term Fab-C also encompasses Fabs comprising the natural amino acids of the hinge region, including the natural cysteine at the C-terminus. In some embodiments, the Fab comprises engineered cysteines (ie, the Fab may be THIOMAB).
“Fab-C”是指具有C-末端半胱氨酸的Fab，所述半胱氨酸可以是在该残基位置出现的天然半胱氨酸(诸如来自铰链区的半胱氨酸)，或者可以是添加到C-末端的半胱氨酸(其不对应于天然半胱氨酸)。抗-因子D抗体包括但不限于AFD.C抗体，其中“C”表示抗体是具有C-末端半胱氨酸的Fab。非限制性的示例性Fab-C重链恒定区包括SEQ ID NO:56、57、59、60、61、62、68和74的序列。"Fab-C" refers to a Fab with a C-terminal cysteine, which may be the native cysteine (such as the cysteine from the hinge region) occurring at this residue position, Or it may be a cysteine (which does not correspond to the natural cysteine) added to the C-terminus. Anti-Factor D antibodies include, but are not limited to, AFD.C antibodies, where "C" indicates that the antibody is a Fab with a C-terminal cysteine. Non-limiting exemplary Fab-C heavy chain constant regions include the sequences of SEQ ID NO:56, 57, 59, 60, 61, 62, 68 and 74.
“Fab-SH”是指具有游离硫醇基团的Fab。在一些实施方案中，游离硫醇基团位于Fab的C-末端的最后10个氨基酸。Fab-C抗体通常也是Fab-SH抗体。另外的非限制性的示例性Fab-SH重链恒定区具有SEQ ID NO:58的氨基酸序列。通常，包含工程改造的半胱氨酸的Fab(即，其为THIOMAB的Fab)是Fab-SH。"Fab-SH" refers to a Fab with a free thiol group. In some embodiments, the free thiol group is located in the last 10 amino acids of the C-terminus of the Fab. Fab-C antibodies are usually also Fab-SH antibodies. Another non-limiting exemplary Fab-SH heavy chain constant region has the amino acid sequence of SEQ ID NO:58. Typically, the Fab comprising engineered cysteines (ie, the Fab which is THIOMAB) is Fab-SH.
术语“可变区”或“可变结构域”是指参与抗体结合至抗原的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域(分别是VH和VL)通常具有相似的结构，各结构域包含四个保守的框架区(FR)和三个高变区(HVR)。(参见，例如Kindt等人KubyImmunology,第6版,W.H.Freeman&Co.,第91页(2007))。单个VH结构域或VL结构域可以足以赋予抗原结合特异性。另外，结合特定抗原的抗体可以使用来自结合该抗原的抗体的VH或VL结构域进行分离，以分别筛选互补VL结构域或VH结构域的文库。参见，例如Portolano等人,J.Immunol.150:880-887(1993)；Clarkson等人,Nature 352:624-628(1991)。The term "variable region" or "variable domain" refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). (See, eg, Kindt et al. Kuby Immunology, 6th Ed., W.H. Freeman & Co., p. 91 (2007)). A single VH domain or VL domain may be sufficient to confer antigen binding specificity. Additionally, antibodies that bind a particular antigen can be isolated using the VH or VL domains from antibodies that bind that antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
术语“可变”是指这样的事实，即在抗体之间可变结构域的某些部分在序列上差异很大并且被用于各特定抗体对其特定抗原的结合和特异性。然而，可变性在抗体的可变结构域上并不是均匀分布的。其集中于在轻链和重链可变结构域中的三个被称为高变区的区段。可变结构域的更高度保守的部分被称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR，其大多采取β折叠构型，通过三个高变区连接，所述三个高变区形成环连接，并且在一些情况下形成β折叠结构的部分。每条链中的高变区通过FR非常接近地保持在一起，并与另一条链的高变区一起促成抗体的抗原结合位点的形成(参见Kabat等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。恒定结构域不直接参与抗体与抗原的结合，但是展现出多种效应子功能，诸如在抗体依赖性细胞毒性(ADCC)中抗体的参与。The term "variable" refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable domains of antibodies. It is centered on three segments called hypervariable regions in the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FRs, mostly in a beta-sheet configuration, connected by three hypervariable regions that form loops connecting and, in some cases, forming Part of the beta sheet structure. The hypervariable regions in each chain are held together in close proximity by FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, p. 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the involvement of the antibody in antibody-dependent cellular cytotoxicity (ADCC).
用木瓜蛋白酶消化抗体产生称作“Fab”片段的两个相同的抗原结合片段(各自具有单个抗原结合位点)和一个残余“Fc”片段，其名称反映了它易于结晶的能力。木瓜蛋白酶处理产生具有两个抗原结合位点并且仍然能够交联抗原的F(ab′)2片段。Digestion of antibodies with papain yields two identical antigen-binding fragments called "Fab" fragments (each with a single antigen-combining site) and a residual "Fc" fragment, whose name reflects its ability to readily crystallize. Papain treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
Fab片段还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)。Fab’片段(包括Fab-C)与Fab片段的不同之处在于在重链CH1结构域的羧基末端添加了几个残基，包括来自抗体铰链区的一个或多个半胱氨酸。具有游离硫醇基团的抗体片段可以表示为具有“-SH”。Fab’-SH(包括Fab-C-SH)是其中恒定结构域的至少一个半胱氨酸残基携带游离硫醇基团的Fab的名称。F(ab′)2抗体片段最初是作为在它们之间具有铰链半胱氨酸的Fab’片段对而产生的。抗体片段的其它化学偶联也是已知的。The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments (including Fab-C) differ from Fab fragments by the addition of several residues at the carboxy-terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region. Antibody fragments with free thiol groups can be indicated as having "-SH". Fab'-SH (including Fab-C-SH) is the designation for a Fab in which at least one cysteine residue of the constant domain bears a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
“Fv”是含有完整抗原识别和抗原结合位点的最小抗体片段。该区由一个重链可变结构域和一个轻链可变结构域以紧密的、非共价缔合的二聚体组成。在这种构型中，每个可变结构域的三个高变区相互作用从而限定在VH-VL二聚体的表面上的抗原结合位点。总而言之，六个高变区将抗原结合特异性赋予抗体。然而，即使单个可变结构域(或只包含对抗原具有特异性的三个高变区的Fv的一半)也具有识别和结合抗原的能力，虽然亲和性低于完整结合位点。"Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Taken together, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only the three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.
如本文所使用的，术语“高变区”或“HVR”是指抗体可变结构域中序列上高度可变和/或形成结构确定的环(“高变环”)的各区域。通常，天然的四链抗体包含六个HVR；三个在VH中(H1、H2、H3)和三个在VL中(L1、L2、L3)。HVR通常包含来自高变环和/或来自“互补决定区”(CDR)的氨基酸残基，后者具有最高序列可变性和/或参与抗原识别。据信HVR-H3在赋予抗体优良的特异性方面发挥独特的作用。参见，例如，Xu等人(2000)Immunity 13:37-45；Johnson和Wu(2003)于Methods in Molecular Biology 248:1-25(Lo编,Human Press,Totowa,N.J.)。“框架区”或“FR”残基是不同于如本文所限定的高变区残基的那些可变结构域残基。如本文所使用的，HVR区包含位于位置24-36(对于L1)、46-56(对于L2)、89-97(对于L3)、26-35B(对于H1)、47-65(对于H2)和93-102(对于H3)内的任意数目的残基。因此，HVR包括在前述位置中的残基：As used herein, the term "hypervariable region" or "HVR" refers to regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Typically, native four-chain antibodies contain six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), which have the highest sequence variability and/or are involved in antigen recognition. It is believed that HVR-H3 plays a unique role in conferring superior specificity to the antibody. See, eg, Xu et al. (2000) Immunity 13:37-45; Johnson and Wu (2003) in Methods in Molecular Biology 248:1-25 (ed. Lo, Human Press, Totowa, N.J.). "Framework region" or "FR" residues are those variable domain residues that are different from the hypervariable region residues as defined herein. As used herein, the HVR region comprises the cells located at positions 24-36 (for L1), 46-56 (for L2), 89-97 (for L3), 26-35B (for H1), 47-65 (for H2) and any number of residues within 93-102 (for H3). Thus, HVRs include residues in the aforementioned positions:
A)24-34(L1)，50-52(L2)，91-96(L3)，26-32(H1)，53-55(H2)，和96-101(H3)(Chothia和Lesk,J.Mol.Biol.196:901-917(1987)；A) 24-34(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J . Mol. Biol. 196:901-917 (1987);
B)L1的24-34，L2的50-56，L3的89-97，H1的31-35B，H2的50-65，和H3的95-102(Kabat等人,Sequences of Proteins of Immunological Interest,第5版,PublicHealth Service,National Institutes of Health,Bethesda,MD(1991)。B) 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).
C)30-36(L1)，46-55(L2)，89-96(L3)，30-35(H1)，47-58(H2)，93-100a-j(H3)(MacCallum等人J.Mol.Biol.262:732-745(1996)。C) 30-36(L1), 46-55(L2), 89-96(L3), 30-35(H1), 47-58(H2), 93-100a-j(H3) (MacCallum et al. J . Mol. Biol. 262:732-745 (1996).
高变区可以包含以下“延长的高变区”：在VL中的24-36或24-34(L1)，46-56或50-56(L2)以及89-97(L3)，和在VH中的26-35B(H1)，50-65、47-65或49-65(H2)以及93-102、94-102或95-102(H3)。对于这些定义中的每个，可变结构域残基根据Kabat等人(见上)进行编号。The hypervariable regions may comprise the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL, and 89-97 (L3) in the VH 26-35B(H1), 50-65, 47-65 or 49-65(H2) and 93-102, 94-102 or 95-102(H3) among them. For each of these definitions, variable domain residues are numbered according to Kabat et al. (supra).
除了VH中的CDR1以外，CDR通常包含形成高变环的氨基酸残基。CDR还包含“特异性决定残基”或“SDR”，其是接触抗原的残基。SDR被包含在CDR的被称为简短CDR(abbreviated-CDR)或a-CDR的区域内。示例性a-CDR(a-CDR-L1、a-CDR-L2、a-CDR-L3、a-CDR-H1、a-CDR-H2和a-CDR-H3)出现于L1的氨基酸残基31-34处、L2的氨基酸残基50-55处、L3的氨基酸残基89-96处、H1的氨基酸残基31-35B处、H2的氨基酸残基50-58处和H3的氨基酸残基95-102处。(参见Almagro和Fransson,Front.Biosci.13:1619-1633(2008))。With the exception of CDR1 in VH, the CDRs generally contain amino acid residues that form hypervariable loops. CDRs also comprise "specificity determining residues" or "SDRs", which are the residues that contact the antigen. The SDR is included in a region of the CDR called an abbreviated-CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residue 31 of L1 -34, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2, and amino acid residue 95 of H3 -102 places. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)).
参比抗体(也称为“起始抗体”或“亲本抗体”)的“抗体变体”或“修饰的抗体”是包含与参比/起始抗体的氨基酸序列不同的氨基酸序列的抗体，其中参比抗体的一个或多个氨基酸残基已被修饰。通常，抗体变体将与参比抗体具有至少80％序列同一性，优选地至少90％序列同一性，更优选地至少95％序列同一性，并且最优选地至少98％序列同一性。百分比序列同一性例如通过Fitch等人,Proc.Natl.Acad.Sci.USA,80:1382-1386(1983),Needleman等人,J.Mol.Biol.,48:443-453(1970)描述的算法版本，在将参比抗体的序列与候选抗体变体比对以提供最大同源性后确定。同一性或相似性在本文中被限定为，在对序列进行比对并且在需要的情况下引入间隙(gap)以实现最大百分比序列同一性后，候选变体序列中与亲本抗体残基相同(即相同的残基)或相似(即来自基于共有侧链性质的相同组的氨基酸残基，见下)的氨基酸残基的百分比。可以通过将适合的核苷酸改变引入到编码抗体的DNA或者通过肽合成来制备抗体的氨基酸序列变体。这种变体包括，例如，从目的抗体的氨基酸序列内的残基缺失，和/或向目的抗体的氨基酸序列内的残基插入，和/或目的抗体的氨基酸序列内的残基置换。可以产生缺失、插入和置换的任意组合以实现最终构建体，条件是所述最终构建体拥有所需特征。氨基酸变化也可以改变抗体的翻译后加工，诸如改变糖基化位点的数量或位置。用于产生抗体的抗体序列变体的方法类似于例如美国专利号5,534,615中描述的用于产生多肽的氨基酸序列变体的那些方法，所述专利通过引用清楚地结合于此。An "antibody variant" or "modified antibody" of a reference antibody (also referred to as a "starting antibody" or "parent antibody") is an antibody comprising an amino acid sequence that differs from that of the reference/starting antibody, wherein One or more amino acid residues of the reference antibody have been modified. Typically, antibody variants will have at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 98% sequence identity to the reference antibody. Percent sequence identity is described, for example, by Fitch et al, Proc. Algorithmic version, determined after aligning the sequence of a reference antibody to a candidate antibody variant to provide maximum homology. Identity or similarity is defined herein as the residues in the candidate variant sequence that are identical to the parent antibody after aligning the sequences and introducing gaps where necessary to achieve the maximum percent sequence identity ( ie identical residues) or similar (ie amino acid residues from the same group based on shared side chain properties, see below) amino acid residues. Amino acid sequence variants of antibodies can be prepared by introducing appropriate nucleotide changes into the DNA encoding the antibody or by peptide synthesis. Such variants include, for example, deletions of residues from, and/or insertions into, and/or substitutions of residues within the amino acid sequence of the antibody of interest. Any combination of deletions, insertions and substitutions can be made to achieve the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter the post-translational processing of the antibody, such as altering the number or location of glycosylation sites. Methods for generating antibody sequence variants of antibodies are similar to those for generating amino acid sequence variants of polypeptides described, for example, in US Patent No. 5,534,615, expressly incorporated herein by reference.
如果包括抗体在内的蛋白质基本上保持完整的构象结构和生物学活性，则称其为“稳定的”。多种用于测量蛋白质稳定性的分析技术是本领域中可获得的并且综述于例如Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed.,Marcel Dekker,Inc.,New York,N.Y.,Pubs.(1991)和Jones(1993)Adv.Drug Delivery Rev.10:29-90中。具有“改善的稳定性”的抗体变体是指与起始参比抗体相比更稳定的抗体变体。优选地，具有改善的稳定性的抗体变体是天然(野生型)抗体的变体，其中特定的氨基酸残基被改变以用于改善天然抗体的物理稳定性、和/或化学稳定性、和/或生物学活性、和/或降低其免疫原性。Walsh(2000)Nat.Biotech.18:831-3。Proteins, including antibodies, are said to be "stable" if they retain substantially their intact conformational structure and biological activity. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones (1993) Adv. Drug Delivery Rev. 10:29-90. An antibody variant with "improved stability" refers to an antibody variant that is more stable compared to the starting reference antibody. Preferably, the antibody variant with improved stability is a variant of a native (wild type) antibody in which specific amino acid residues have been altered for improving the physical stability, and/or chemical stability, of the native antibody, and /or biological activity, and/or reduce its immunogenicity. Walsh (2000) Nat. Biotech. 18:831-3.
术语“异构化”通常是指这样一种化学过程，通过所述化学过程化学化合物被转化为其异构形式，即，具有相同的化学组成但是具有不同的结构或构型并且因此通常具有不同的物理和化学性质的形式。本文中具体使用的是天冬氨酸异构化，其是这样的过程，其中多肽的一个或多个天冬氨酸(D或Asp)残基被转化为异天冬氨酸残基。Geiger和Clarke(1987)J.Biol.Chem.262:785-94。The term "isomerization" generally refers to the chemical process by which a chemical compound is converted into its isomeric form, that is, having the same chemical composition but a different structure or configuration and thus often a different form of physical and chemical properties. Specifically used herein is aspartic acid isomerization, which is a process in which one or more aspartic acid (D or Asp) residues of a polypeptide are converted to isoaspartic acid residues. Geiger and Clarke (1987) J. Biol. Chem. 262:785-94.
术语“脱酰胺”通常是指这样的化学反应，其中酰胺官能团被从有机化合物除去。本文中具体使用的是天冬酰胺脱酰胺，其是这样的过程，其中多肽的一个或多个天冬酰胺(N或Asn)残基被转化为天冬氨酸(D或Asp)，即中性酰胺侧链被转化为整体具有酸性的残基。Xie和Schowen(1999)J.Pharm.Sci.88:8-13。The term "deamidation" generally refers to a chemical reaction in which an amide function is removed from an organic compound. Specifically used herein is asparagine deamidation, which is a process in which one or more asparagine (N or Asn) residues of a polypeptide are converted to aspartic acid (D or Asp), i.e., The neutral amide side chain is converted to an overall acidic residue. Xie and Schowen (1999) J. Pharm. Sci. 88:8-13.
氨基酸残基“倾向”于某些鉴定的物理或化学过程(例如，异构化或脱酰胺)是指特定蛋白质分子内被鉴定为具有进行鉴定的过程(诸如异构化或脱酰胺)的倾向的那些残基。它们的倾向通常通过它们在蛋白质的一级和/或构象结构内的相对位置确定。例如，已经证明，Asp-XXX基序(其中XXX可以是Asp、Gly、His、Ser或Thr)中的第一个Asp由于其相邻残基的参与而倾向于Asp异构化，其中相同蛋白质内的一些其它Asp可能不具有这种倾向。用于在具体蛋白质分子内鉴定残基对某些过程的倾向性的测定是本领域中已知的。参见，例如，Cacia等人(1996)Biochem.35:1897-1903。"Propensity" of amino acid residues to certain identified physical or chemical processes (e.g., isomerization or deamidation) refers to an identified tendency within a particular protein molecule to undergo an identified process (such as isomerization or deamidation) those residues. Their propensity is usually determined by their relative position within the protein's primary and/or conformational structure. For example, it has been shown that the first Asp in an Asp-XXX motif (where XXX can be Asp, Gly, His, Ser, or Thr) is prone to Asp isomerization due to the participation of its neighboring residues, where the same protein Some other Asp within may not have this tendency. Assays for identifying the propensity of residues within a particular protein molecule to certain processes are known in the art. See, eg, Cacia et al. (1996) Biochem. 35:1897-1903.
在本发明的抗-因子D抗体的语境中，“活性的”或“活性”或“生物活性”是对抗(部分或完全抑制)因子D的生物活性的能力。因子D拮抗剂的生物活性的一个实例是实现可测量的因子D相关疾病或病症(诸如例如补体相关性眼部病症)的状态(例如病理学)的改善的能力。可以在体外或体内测试中使用相关动物模型或人临床试验来确定活性，所述测试包括结合测定、备选途径溶血测定(例如测量备选途径补体活性或激活的抑制的测定)。"Active" or "activity" or "biological activity" in the context of the anti-Factor D antibodies of the invention is the ability to antagonize (partially or completely inhibit) the biological activity of Factor D. One example of a biological activity of a Factor D antagonist is the ability to achieve a measurable improvement in the state (eg, pathology) of a Factor D-associated disease or disorder, such as, for example, a complement-associated ocular disorder. Activity can be determined in in vitro or in vivo assays using relevant animal models or human clinical assays, including binding assays, alternative pathway hemolysis assays (eg, assays that measure inhibition of alternative pathway complement activity or activation).
术语“补体相关性病症”以最广意义使用并且包括与过度或不受控的补体激活相关的病症。其包括心肺分流术期间的补体激活；由急性心肌梗死、动脉瘤、卒中、出血性休克、挤压伤、多器官衰竭、低血容量性休克、肠缺血或其它引起缺血的事件之后的缺血-再灌注引起的补体激活。补体激活还已经被证明与炎症病症相关，所述炎症病症诸如严重烧伤、内毒素血症、脓毒性休克、成人呼吸窘迫综合征、血液透析；过敏性休克、严重哮喘、血管性水肿、克罗恩病(Crohn’s disease)、镰状细胞性贫血、链球菌感染后肾小球肾炎和胰腺炎。所述病况可以是不良药物反应、药物变态反应、IL-2诱导的血管渗漏综合征或放射摄影造影剂变态反应的结果。其还包括自身免疫疾病，诸如系统性红斑狼疮、重症肌无力、类风湿性关节炎、阿尔茨海默病(Alzheimer’s disease)和多发性硬化。补体激活还与移植排斥相关。补体激活还与眼病相关，所述眼病诸如年龄相关性黄斑变性、糖尿病性视网膜病和其它缺血相关性视网膜病、脉络膜新血管形成(CNV)、葡萄膜炎、糖尿病性黄斑水肿、病理性近视、希佩尔-林道病、眼的组织胞浆菌病、视网膜中央静脉阻塞(CRVO)、角膜新血管形成和视网膜新血管形成。The term "complement-associated disorder" is used in the broadest sense and includes disorders associated with excessive or uncontrolled complement activation. It includes complement activation during cardiopulmonary bypass; following acute myocardial infarction, aneurysm, stroke, hemorrhagic shock, crush injury, multiorgan failure, hypovolemic shock, intestinal ischemia, or other events causing ischemia Activation of complement by ischemia-reperfusion. Complement activation has also been shown to be associated with inflammatory conditions such as severe burns, endotoxemia, septic shock, adult respiratory distress syndrome, hemodialysis; anaphylactic shock, severe asthma, angioedema, Crohn's Crohn's disease, sickle cell anemia, poststreptococcal glomerulonephritis, and pancreatitis. The condition may be the result of an adverse drug reaction, drug allergy, IL-2-induced vascular leak syndrome, or radiographic contrast media allergy. It also includes autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Alzheimer's disease and multiple sclerosis. Complement activation is also associated with transplant rejection. Complement activation is also associated with eye diseases such as age-related macular degeneration, diabetic retinopathy and other ischemia-related retinopathy, choroidal neovascularization (CNV), uveitis, diabetic macular edema, pathological myopia , Hipper-Lindau disease, histoplasmosis of the eye, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization.
术语“补体相关性眼部病症”以最广意义使用并且包括病理学涉及补体(包括经典和旁路途径，并且特别是补体的旁路途径)的所有眼部病症。补体相关性眼部病症包括，但不限于，黄斑变性病(诸如所有阶段的年龄相关性黄斑变性(AMD)，包括干性和湿性(非渗出性和渗出性)形式)，脉络膜新血管形成(CNV)，葡萄膜炎，糖尿病性和其它缺血相关性视网膜病，以及其它眼内新生血管性疾病，诸如糖尿病性黄斑水肿、病理性近视、希佩尔-林道病、眼的组织胞浆菌病、视网膜中央静脉阻塞(CRVO)，角膜新血管形成和视网膜新血管形成。在一个实例中，补体相关性眼部病症包括年龄相关性黄斑变性(AMD)，包括非渗出性(例如中期干性AMD或地图状萎缩(GA))和渗出性(例如湿性AMD(脉络膜新血管形成(CNV))AMD，糖尿病性视网膜病(DR)，眼内炎和葡萄膜炎。在另外的实例中，非渗出性AMD可以包括硬玻璃疣、软玻璃疣、地图状萎缩和/或色素凝集的存在。在一个实例中，补体相关性眼部病症包括年龄相关性黄斑变性(AMD)，包括早期AMD(例如包括多个小的至一个或多个非渗出性的中等大小的玻璃疣)、中期AMD(例如包括大量的中等玻璃疣至一个或多个大玻璃疣)和晚期AMD(例如包括地图状萎缩或晚期湿性AMD(CNV)。(Ferris等人,AREDS Report No.18；Sallo等人,Eye Res.,34(3):238-40(2009)；Jager等人,New Engl.J.Med.,359(1):1735(2008))。在另外的实例中，中期干性AMD可以包括大的汇合的玻璃疣。在另外的实例中，地图状萎缩可以包括光感受器和/或视网膜色素上皮(RPE)损失。在另外的实例中，地图状萎缩的面积可以是小的或大的和/或可以是在黄斑区域中或在外围视网膜中。在一个实例中，补体相关性眼部病症是中期干性AMD。在一个实例中，补体相关性眼部病症是地图状萎缩。在一个实例中，补体相关性眼部病症是湿性AMD(脉络膜新血管形成(CNV))。The term "complement-associated ocular disorder" is used in the broadest sense and includes all ocular disorders whose pathology involves complement, including the classical and alternative pathways, and in particular the alternative pathway of complement. Complement-associated ocular disorders include, but are not limited to, macular degeneration (such as all stages of age-related macular degeneration (AMD), including dry and wet (non-exudative and exudative) forms), choroidal neovascularization formation (CNV), uveitis, diabetic and other ischemia-related retinopathy, and other intraocular neovascular diseases such as diabetic macular edema, pathological myopia, Hipper-Lindau disease, histiocytosis of the eye Plasma disease, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization. In one example, the complement-associated ocular disorder includes age-related macular degeneration (AMD), including non-exudative (eg, intermediate dry AMD or geographic atrophy (GA)) and exudative (eg, wet AMD (choroidal atrophy) Neovascularization (CNV)) AMD, diabetic retinopathy (DR), endophthalmitis, and uveitis. In additional examples, non-exudative AMD can include hard drusen, soft drusen, geographic atrophy, and and/or the presence of pigment agglutination. In one example, the complement-associated ocular disorder includes age-related macular degeneration (AMD), including early AMD (e.g., including multiple small to one or more non-exudative medium-sized drusen), intermediate AMD (eg, including numerous medium drusen to one or more large drusen), and advanced AMD (eg, including geographic atrophy or advanced wet AMD (CNV). (Ferris et al., AREDS Report No. 18; Sallo et al., Eye Res., 34(3):238-40 (2009); Jager et al., New Engl.J.Med., 359(1):1735(2008)). In additional examples , mid-stage dry AMD can include large confluent drusen. In another example, geographic atrophy can include photoreceptor and/or retinal pigment epithelium (RPE) loss. In another example, the area of geographic atrophy can be is small or large and/or may be in the macular area or in the peripheral retina. In one example, the complement-associated ocular disorder is intermediate dry AMD. In one example, the complement-associated ocular disorder is Geographic atrophy. In one example, the complement-associated ocular disorder is wet AMD (choroidal neovascularization (CNV)).
“治疗(treatment)”(及其语法上的变体，诸如“treat”或“treating”)是以阻止病症的病理学的发展或改变病症的病理学为目的而进行的干预。因此，“治疗”是指治疗性治疗和预防性或防预性的措施。需要治疗的那些包括已经患有病症的那些以及要预防病症的那些。所需的治疗效果包括，但不限于，防止疾病出现或复发，减轻症状，减小疾病的任何直接或间接病理学后果，降低病情进展速率，改善或缓和疾病状态，以及缓解或预后改善。在一些实施方案中，本发明的缀合物是用来延缓疾病发展或用来减慢疾病的进展。在免疫相关疾病的治疗中，治疗剂可以直接改变免疫反应的组分的反应程度，或者通过其它治疗剂(例如抗生素、抗真菌剂、消炎剂、化疗剂等)使疾病对治疗更敏感。"Treatment" (and its grammatical variants, such as "treat" or "treating") is an intervention with the purpose of arresting the development of, or altering, the pathology of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Desired therapeutic effects include, but are not limited to, prevention of onset or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the conjugates of the invention are used to delay the development of a disease or to slow the progression of a disease. In the treatment of immune-related diseases, therapeutic agents can directly alter the degree of response of components of the immune response, or render the disease more sensitive to treatment through other therapeutic agents (eg, antibiotics, antifungals, anti-inflammatory agents, chemotherapeutics, etc.).
疾病(诸如补体相关性眼部病症)的“病理学”包括危及患者健康的所有现象。这包括，但不限于，异常或不受控的细胞生长(嗜中性粒细胞、嗜酸性粒细胞、单核细胞、淋巴细胞)、抗体产生、自身抗体产生、补体产生、干扰邻近细胞的正常功能、以异常水平释放细胞因子或其它分泌性产物、抑制或加重任何炎症反应或免疫反应、炎症细胞(嗜中性粒细胞、嗜酸性粒细胞、单核细胞、淋巴细胞)渗入到细胞空间中等。The "pathology" of a disease, such as a complement-associated ocular disorder, includes all phenomena that endanger the health of the patient. This includes, but is not limited to, abnormal or uncontrolled cell growth (neutrophils, eosinophils, monocytes, lymphocytes), antibody production, autoantibody production, complement production, interference with normal function, release of cytokines or other secretory products at abnormal levels, suppression or exacerbation of any inflammatory or immune response, infiltration of inflammatory cells (neutrophils, eosinophils, monocytes, lymphocytes) into the cellular space, etc. .
如本文所使用的，术语“哺乳动物”是指被分类为哺乳动物的任何动物，包括但不限于人、高等灵长类、家养和农场动物，以及动物园动物、竞技动物或宠物，诸如马、猪、牛、狗、猫和雪貂等。在本发明的一些实施方案中，哺乳动物是人。As used herein, the term "mammal" refers to any animal classified as a mammal, including but not limited to humans, higher primates, domestic and farm animals, as well as zoo animals, sport animals or pets such as horses, Pigs, cows, dogs, cats and ferrets etc. In some embodiments of the invention, the mammal is a human.
与一种或多种另外的治疗剂“组合”施用包括同时(并行)施用和以任何次序的连续施用。Administration "in combination" with one or more additional therapeutic agents includes both simultaneous (concurrent) administration and sequential administration in any order.
“治疗有效量”是实现可测量的目标疾病或病症(诸如例如补体相关性眼部病症)的状态(例如病理学)的改善所需要的“因子D拮抗剂”的量。A "therapeutically effective amount" is the amount of "Factor D antagonist" required to achieve a measurable amelioration of the state (eg, pathology) of the target disease or disorder, such as, for example, a complement-associated ocular disorder.
“氨基酸置换”是指将预先确定的氨基酸序列中的至少一个现有的氨基酸残基替换成另一种不同的“替代”氨基酸残基。替代残基可以是“天然存在的氨基酸残基”(即，由遗传密码编码的)并且选自由以下组成的组：丙氨酸(ala)；精氨酸(Arg)；天冬酰胺(Asn)；天冬氨酸(Asp)；半胱氨酸(Cys)；谷氨酰胺(Gln)；谷氨酸(Glu)；甘氨酸(Gly)，组氨酸(His)；异亮氨酸(Ile)；亮氨酸(Leu)；赖氨酸(Lys)；甲硫氨酸(Met)；苯丙氨酸(Phe)；脯氨酸(Pro)；丝氨酸(Ser)；苏氨酸(Thr)；色氨酸(Trp)；酪氨酸(Tyr)；和缬氨酸(Val)。用一个或多个非天然存在的氨基酸残基进行的置换也被包括在本文中氨基酸置换的定义中。“非天然存在的氨基酸残基”是指不同于以上列出的那些天然存在的氨基酸残基的残基，其能够共价结合多肽链中的邻近氨基酸残基。非天然存在的氨基酸残基的实例包括正亮氨酸、鸟氨酸、正缬氨酸、高丝氨酸和其他氨基酸残基类似物如Ellman等人,Meth.Enzym,202:301-336(1991)中所述的那些。为了产生此种非天然存在的氨基酸残基，可以使用Noren等人,Science,244:182(1989)和Ellman等人(见上)的方法。简言之，这些方法包括化学活化具有非天然存在的氨基酸残基的抑制tRNA，之后体外转录和翻译所述RNA。"Amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different "substitute" amino acid residue. The replacement residue may be a "naturally occurring amino acid residue" (i.e., encoded by the genetic code) and be selected from the group consisting of: alanine (ala); arginine (Arg); asparagine (Asn) ; Aspartic acid (Asp); Cysteine (Cys); Glutamine (Gln); Glutamic acid (Glu); Glycine (Gly), Histidine (His); Isoleucine (Ile) ;Leucine (Leu); Lysine (Lys); Methionine (Met); Phenylalanine (Phe); Proline (Pro); Serine (Ser); Threonine (Thr); Tryptophan (Trp); Tyrosine (Tyr); and Valine (Val). Substitutions with one or more non-naturally occurring amino acid residues are also included within the definition of amino acid substitution herein. "Non-naturally occurring amino acid residue" refers to a residue other than those naturally occurring amino acid residues listed above that is capable of covalently binding to adjacent amino acid residues in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs such as Ellman et al., Meth. Enzym, 202:301-336 (1991) those described in . To generate such non-naturally occurring amino acid residues, the methods of Noren et al., Science, 244:182 (1989) and Ellman et al. (supra) can be used. Briefly, these methods involve chemical activation of a suppressor tRNA with non-naturally occurring amino acid residues, followed by in vitro transcription and translation of the RNA.
“氨基酸插入”是指将至少一个氨基酸结合到预先确定的氨基酸序列中。虽然插入通常由一个或两个氨基酸残基的插入组成，但是本申请预期更大的“肽插入”，例如插入约三至约五个或甚至多达约十个氨基酸残基。插入的残基可以是如以上公开的天然存在的或非天然存在的氨基酸残基。"Amino acid insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. While insertions typically consist of insertions of one or two amino acid residues, the present application contemplates larger "peptide insertions," such as insertions of about three to about five or even up to about ten amino acid residues. The inserted residues may be naturally occurring or non-naturally occurring amino acid residues as disclosed above.
“氨基酸缺失”是指从预先确定的氨基酸序列除去至少一个氨基酸残基。"Amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.
术语“长效递送”、“持续释放”和“受控释放”通常用于描述利用制剂、剂型、装置或其它类型的技术以实现治疗性药物的延长的或延展的释放或生物利用度的递送机制。其可以指向全身体循环或受试者或向受试者中的局部作用位点(包括但不限于细胞、组织、器官、关节、区域等)提供药物的延长的或延展的释放或生物利用度的技术。此外，这些术语可以指用于延长或延展药物从制剂或剂型的释放的技术，或者它们可以指用于延展或延长药物的生物利用度或药物动力学或对受试者的作用持续时间的技术，或者它们可以指用于延展或延长制剂引发的药物动力学效应的技术。“长效制剂”、“持续释放制剂”或“受控释放制剂”是用于提供长效递送的药物制剂、剂型或其它技术。在一些方面，受控释放用于改善药物的局部生物利用度，特别是在眼部递送的情况中，其用于改善眼部停留时间。“增加的眼部停留时间”是指递送后的时间段，在这期间递送的眼部药物在质量(活性)和数量(有效量)方面都保持有效。除了高剂量和受控释放以外或作为高剂量和受控释放的替代，药物可以被翻译后修饰(诸如经由PEG化)，以实现增加的体内半衰期。The terms "long-acting delivery", "sustained release" and "controlled release" are generally used to describe the delivery of a therapeutic drug using a formulation, dosage form, device or other type of technology to achieve prolonged or extended release or bioavailability mechanism. It may provide prolonged or extended release or bioavailability of the drug directed to the general systemic circulation or the subject or to a localized site of action in the subject (including but not limited to cells, tissues, organs, joints, regions, etc.) Technology. Additionally, these terms may refer to techniques used to prolong or prolong the release of a drug from a formulation or dosage form, or they may refer to techniques used to prolong or prolong the bioavailability or pharmacokinetics or duration of action of a drug in a subject , or they can refer to techniques used to extend or prolong the pharmacokinetic effects induced by a formulation. A "depot formulation", "sustained release formulation" or "controlled release formulation" is a pharmaceutical formulation, dosage form or other technology used to provide long-term delivery. In some aspects, controlled release is used to improve the local bioavailability of the drug, particularly in the case of ocular delivery, which is used to improve ocular residence time. "Increased ocular residence time" refers to the period of time after delivery during which the delivered ocular drug remains effective, both in quality (activity) and quantity (effective amount). In addition to or as an alternative to high dose and controlled release, drugs may be post-translationally modified, such as via PEGylation, to achieve increased half-life in vivo.
术语“端口递送系统”是指具有可再填充的储器的用于眼睛的可植入装置，其允许在延长的时间段内递送治疗剂。示例性的端口递送系统描述于例如美国专利申请序列号2010/0174272，以及美国专利号8,277,830；8,399,006；8,795,712；和8,808,727中，所有这些都通过引用结合于此。The term "port delivery system" refers to an implantable device for use in the eye with a refillable reservoir that allows delivery of a therapeutic agent over an extended period of time. Exemplary port delivery systems are described, for example, in US Patent Application Serial No. 2010/0174272, and US Patent Nos. 8,277,830; 8,399,006; 8,795,712; and 8,808,727, all of which are incorporated herein by reference.
当在本文中使用时，术语“多元醇”在广义上是指多羟基醇化合物。多元醇可以是例如任何水溶性聚(氧化烯)聚合物，并且能够具有直链或支链。优选的多元醇包括在一个或多个羟基位置处被化学基团(诸如，具有一至四个碳的烷基)取代的那些多元醇。典型地，多元醇是聚(亚烷基二醇)，优选聚乙二醇(PEG)。然而，本领域技术人员认识到可以使用本文描述的用于PEG的缀合技术来利用其它多元醇，诸如例如聚(丙二醇)和聚乙二醇-聚丙二醇共聚物。本发明公开内容的多元醇包括本领域熟知的那些和公开可获得的那些，诸如来自可商购的来源的那些。As used herein, the term "polyol" refers broadly to polyhydric alcohol compounds. The polyol can be, for example, any water-soluble poly(oxyalkylene) polymer, and can have straight or branched chains. Preferred polyols include those substituted at one or more hydroxyl positions with chemical groups such as alkyl groups having one to four carbons. Typically, the polyol is a poly(alkylene glycol), preferably polyethylene glycol (PEG). However, those skilled in the art recognize that other polyols such as, for example, poly(propylene glycol) and polyethylene glycol-polypropylene glycol copolymers can be utilized using the conjugation techniques described herein for PEG. The polyols of the present disclosure include those well known in the art and those publicly available, such as those from commercially available sources.
根据其最宽泛的定义，术语“缀合”在本文中用于意指结合或连接在一起。当分子如同结合在一起而起作用或操作时，分子是“缀合的”。在特别的实施方案中，“缀合物”是指与多臂多元醇共价结合的抗体(例如，如本文详述的抗体片段)。According to its broadest definition, the term "conjugate" is used herein to mean bound or linked together. Molecules are "conjugated" when they function or operate as if bound together. In particular embodiments, "conjugate" refers to an antibody (eg, an antibody fragment as detailed herein) covalently bound to a multi-armed polyol.
“小孔径针”或“窄孔径针”是指用于注射流体组合物的约30、29、28、27、26、25、24、23或22号针或更高规格的针，诸如30号针。在一些实施方案中，小孔径针具有标准尺寸的壁。在另一个实施方案中，小孔径针具有薄壁，这对于粘稠溶液可能是优选的。"Small bore needle" or "narrow bore needle" means a needle of about 30, 29, 28, 27, 26, 25, 24, 23 or 22 gauge or higher gauge, such as 30 gauge, for injecting a fluid composition Needle. In some embodiments, small bore needles have walls of standard dimensions. In another embodiment, small bore needles have thin walls, which may be preferred for viscous solutions.
相对于参比多肽序列的“百分比(％)氨基酸序列同一性”定义为在对序列进行比对并且在需要的情况下引入空位以实现最大百分比的序列同一性并且不考虑任何保守性置换作为序列同一性的一部分之后，候选序列中与参比多肽序列中的氨基酸残基相同的氨基酸残基的百分比。为了确定氨基酸序列同一性百分比的比对可以以本领域能力范围内的多种方式实现，例如，使用可公开获得的计算机软件诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适当参数，包括实现正在比较的全长序列范围内最大比对所需要的任何算法。然而，出于本文目的，使用序列比较计算机程序ALIGN-2产生氨基酸序列同一性％值。ALIGN-2序列比较计算机程序由Genentech,Inc.创作，并且源代码已经随用户文档提交至U.S.Copyright Office,Washington D.C.,20559(美国版权办公室华盛顿特区20559)，其中它以美国版权登记号TXU510087登记。ALIGN-2程序从Genentech,Inc.,South San Francisco(南旧金山),California(加利福尼亚州)可公开获得或可以从源代码汇编。应当将ALIGN-2程序汇编用以在UNIX操作系统(包括数字式UNIX V4.0D)上使用。全部序列比较参数通过ALIGN-2程序设定并且不变动。"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the sequence after aligning the sequences and introducing gaps where necessary to achieve the maximum percent sequence identity and not taking into account any conservative substitutions as sequences Following a portion of identity, the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California or can be assembled from source code. The ALIGN-2 program should be assembled for use on UNIX operating systems (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
在使用ALIGN-2进行氨基酸序列比较的情况下，如下计算给定的氨基酸序列A与、同或针对给定的氨基酸序列B的％氨基酸序列同一性(这可以备选地描述为给定的氨基酸序列A具有或包含与、同或针对给定的氨基酸序列B的某一％氨基酸序列同一性)：In the case of amino acid sequence comparisons using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to, with or against a given amino acid sequence B (which can alternatively be described as Sequence A has or comprises a certain % amino acid sequence identity to, the same as, or to a given amino acid sequence B):
100乘以分数X/Y，100 times fraction X/Y,
其中X是通过序列比对程序ALIGN-2在该程序的A和B比对中评定为相同匹配的氨基酸残基的数目，并且其中Y是B中氨基酸残基的总数。将可以理解的是，在氨基酸序列A的长度不等于氨基酸序列B的长度时，A相对于B的％氨基酸序列同一性将不等于B相对于A的％氨基酸序列同一性。除非另外特别声明，否则本文所用的全部％氨基酸序列同一性值如紧接前段中所述的使用ALIGN-2计算机程序获得。where X is the number of amino acid residues assessed as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A with respect to B will not be equal to the % amino acid sequence identity of B with respect to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.
术语“药物制剂”是指这样的制备物，其处于这样的形式从而允许包含于其中的活性成分的生物活性有效，并且其不含有对于将施用该制剂的受试者不可接受地有毒的额外组分。The term "pharmaceutical formulation" refers to a preparation which is in such a form as to allow the biological activity of the active ingredient contained therein to be effective and which does not contain additional components which are unacceptably toxic to the subject to which the formulation will be administered. point.
“药用载体”是指除活性成分之外，药物制剂中对受试者无毒的成分。药用载体包括但不限于缓冲液、赋形剂、稳定剂或防腐剂。"Pharmaceutically acceptable carrier" refers to ingredients in pharmaceutical preparations other than the active ingredient that are non-toxic to the subject. Pharmaceutical carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
与一种或多种其它药物“同时”施用的药物在相同的治疗周期期间施用，在与一种或多种其它药物的治疗的同一天施用，和任选地在与一种或多种其它药物的同一时间施用。A drug that is administered "simultaneously" with one or more other drugs is administered during the same treatment cycle, on the same day of treatment as one or more other drugs, and optionally on the same day as one or more other drugs Drugs are administered at the same time.
抗-因子D抗体及其变体Anti-factor D antibodies and variants thereof
在一些方面，本发明涉及包括一个或多个抗-因子D抗体或其变体的缀合物的制备和使用。适用于形成本发明的缀合物的抗-因子D抗体及其变体描述于美国专利申请序列号14/700853(2015年4月30日提交)中，其通过引用整体结合于此。In some aspects, the invention relates to the preparation and use of conjugates comprising one or more anti-Factor D antibodies or variants thereof. Anti-Factor D antibodies and variants thereof suitable for use in forming conjugates of the invention are described in US Patent Application Serial No. 14/700853 (filed April 30, 2015), which is hereby incorporated by reference in its entirety.
在一些方面，形成用于产生用于本发明的缀合物的变体的碱基的亲本参比抗-因子D抗体是人源化抗-因子D抗体。用于将非人抗体人源化的方法是本领域公知的。通常，人源化抗体具有从非人来源引入其中的一个或多个氨基酸残基。这些非人氨基酸残基通常被称为“进口”残基，其典型地取自“进口”可变结构域。人源化可以基本上遵循Winter及同事(Jones等人(1986)Nature 321:522-525；Riechmann等人(1988)Nature 332:323-327；Verhoeyen等人(1988)Science 239:1534-1536)的方法，通过用啮齿动物CDR或CDR序列置换人抗体的相应序列进行。因此，这种”人源化”抗体是嵌合抗体(美国专利号4,816,567)，其中基本上少于完整人可变结构域被来自非人物种的相应序列置换。实际上，人源化抗体典型地是这样的人抗体，其中一些CDR残基和可能的一些FR残基被来自啮齿动物抗体中的类似位点的残基置换。In some aspects, the parental reference anti-Factor D antibody that forms the bases used to generate the variants used in the conjugates of the invention is a humanized anti-Factor D antibody. Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can essentially follow Winter and colleagues (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536) method by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Thus, such "humanized" antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
在欲将抗体用于人治疗用途时，在一些情况下，用于制备人源化抗体的人可变结构域(轻链和重链两者)的选择可能对于降低抗原性和/或HAMA反应(人抗小鼠抗体)是重要的。减少或消除HAMA反应通常是临床开发合适的治疗剂的重要方面。参见，例如，Khaxzaeli等人(1988)J.Natl.Cancer Inst 80:937；Jaffers等人(1986)Transplantation 41:572；Shawler等人(1985)J.Immunol.135:1530；Sears等人(1984)J.Biol.Response Mod.3:138；Miller等人(1983)Blood 62:988；Hakimi等人(1991)J.Immunol.147:1352；Reichmann等人(1988)Nature 332:323；Junghans等人(1990)Cancer Res.50:1495。如本文所述，在一些方面，本发明提供缀合物，其包含被人源化从而减少或消除了HAMA反应的抗体。这些抗体的变体可以进一步使用本领域已知的常规方法获得，所述方法中的一些在下文中被进一步描述。根据所谓的“最佳-配合(best-fit)”方法，针对已知的人可变结构域序列的整个文库来筛选啮齿动物抗体的可变结构域的序列。鉴定与啮齿动物的V结构域序列最接近的人V结构域序列并且其中的人框架区(FR)被接受用于人源化抗体(Sims等人(1993)J.Immunol.151:2296；Chothia等人(1987)J.Mol.Biol.196:901)。另一种方法使用来源于所有特定轻链或重链亚组的人抗体的共有序列的特定框架区。相同的框架可以用于若干不同的人源化抗体(Carter等人(1992)Proc.Natl.Acad.Sci.USA 89:4285；Presta等人(1993)J.Immunol.151:2623)。When antibodies are intended for human therapeutic use, in some cases the selection of human variable domains (both light and heavy chains) used to prepare humanized antibodies may be important in reducing antigenicity and/or HAMA response. (human anti-mouse antibody) is important. Reduction or elimination of HAMA responses is often an important aspect of clinical development of appropriate therapeutics. See, eg, Khaxzaeli et al. (1988) J. Natl. Cancer Inst 80:937; Jaffers et al. (1986) Transplantation 41:572; Shawler et al. (1985) J. Immunol.135:1530; Sears et al. (1984) ) J.Biol.Response Mod.3:138; Miller et al. (1983) Blood 62:988; Hakimi et al. (1991) J.Immunol.147:1352; Reichmann et al. (1988) Nature 332:323; Junghans et al. Man (1990) Cancer Res. 50:1495. As described herein, in some aspects, the invention provides conjugates comprising an antibody that has been humanized to reduce or eliminate the HAMA response. Variants of these antibodies can further be obtained using conventional methods known in the art, some of which are further described below. According to the so-called "best-fit" method, the sequences of the variable domains of rodent antibodies are screened against the entire library of known human variable domain sequences. The human V domain sequence that is closest to the rodent V domain sequence was identified and the human framework regions (FR) in it were accepted for use in humanized antibodies (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA 89:4285; Presta et al. (1993) J. Immunol. 151:2623).
例如，来自如本文描述的抗体的氨基酸序列可以充当用于框架和/或高变序列的多样化的起始(亲本)序列。选择的与起始高变序列相连的框架序列在本文中被称为受体人框架。虽然受体人框架可以来自或来源于人免疫球蛋白(其VL和/或VH区)，但是当这种框架被证明在人类患者中具有最小免疫原性或没有免疫原性时，受体人框架可以来自或来源于人共有框架序列。出于本文目的，“受体人框架”是这样的框架，其包含来源于人免疫球蛋白框架或来自人共有框架的VL或VH框架的氨基酸序列。“来源于”人免疫球蛋白框架或人共有框架的受体人框架可以包含其相同的氨基酸序列，或可以含有预先存在的氨基酸序列改变。当存在预先存在的氨基酸改变时，优选地存在不超过5个并且优选地4以下或3个以下预先存在的氨基酸改变。在一些实施方案中，VH受体人框架在序列上与VH人免疫球蛋白框架序列或人共有框架序列相同。在一些实施方案中，VL受体人框架在序列上与VL人免疫球蛋白框架序列或人共有框架序列相同。“人共有框架”是这样的框架，其代表在人免疫球蛋白VL或VH框架序列的选择中最常出现的氨基酸残基。通常，人免疫球蛋白VL或VH框架序列的选项来自可变结构域序列的亚组。通常，所述序列亚组是如在Kabat等人中的亚组。在一些实施方案中，对于VL，所述亚组是如在Kabat等人中的亚组κI。在一些实施方案中，对于VH，所述亚组是如在Kabat等人中的亚组III。For example, amino acid sequences from antibodies as described herein can serve as starting (parental) sequences for diversification of framework and/or hypervariable sequences. The selected framework sequences linked to the starting hypervariable sequences are referred to herein as the acceptor human framework. Although recipient human frameworks may be derived or derived from human immunoglobulins (the VL and/or VH regions thereof), when such frameworks are shown to be minimally or not immunogenic in human patients, recipient human A framework can be from or derived from a human consensus framework sequence. For the purposes herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or from a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or may contain pre-existing amino acid sequence changes. When there are pre-existing amino acid changes, preferably no more than 5 and preferably less than 4 or less than 3 pre-existing amino acid changes are present. In some embodiments, the VH acceptor human framework is identical in sequence to a VH human immunoglobulin framework sequence or a human consensus framework sequence. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence. A "human consensus framework" is a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH framework sequences is from a subgroup of variable domain sequences. Typically, the subset of sequences is a subset as in Kabat et al. In some embodiments, for VL, the subgroup is subgroup κI as in Kabat et al. In some embodiments, for VH, the subgroup is subgroup III as in Kabat et al.
当受体来源于人免疫球蛋白时，可以在人框架序列的集合中任选地选择基于其与供体框架序列的同源性(通过将供体框架序列与不同人框架序列比对)选择的人框架序列，并且选择同源性最高的框架序列作为受体。受体人框架可以来自或来源于公众数据库中可获得的人抗体种系序列。When the recipient is derived from a human immunoglobulin, one can optionally select among the pool of human framework sequences based on their homology to the donor framework sequence (by aligning the donor framework sequence with a different human framework sequence) Human framework sequence, and select the framework sequence with the highest homology as the acceptor. The acceptor human framework can be derived from or derived from human antibody germline sequences available in public databases.
在一些实施方案中，本文中的人共有框架来自或来源于VH亚组VII和/或VLκ亚组I共有框架序列。In some embodiments, the human consensus framework herein is from or derived from a VH subgroup VII and/or VLκ subgroup I consensus framework sequence.
在一些实施方案中，用于产生抗-因子D抗体的人框架模板可以包含来自包含对于VH链的VI-4.1b+(VH7家族)和对于VL链的DPK4(VκI家族)和JK2的组合的模板的框架序列。In some embodiments, the human framework templates used to generate anti-Factor D antibodies may comprise templates from a combination comprising VI-4.1b+ (VH7 family) for the VH chain and DPK4 (VκI family) and JK2 for the VL chain frame sequence.
虽然受体可以在序列上与选择的人框架序列相同而不论其是来自人免疫球蛋白还是人共有框架，但是本发明预计受体序列相对于所述人免疫球蛋白序列或人共有框架序列可以包含预先存在的氨基酸置换。这些预先存在的置换优选地是最少的；通常是相对于所述人免疫球蛋白序列或共有框架序列的四个、三个、两个或一个氨基酸差异。Although the acceptor may be identical in sequence to a selected human framework sequence, whether from a human immunoglobulin or a human consensus framework, the present invention contemplates that the acceptor sequence may be identical to the human immunoglobulin sequence or human consensus framework sequence. Contains pre-existing amino acid substitutions. These pre-existing substitutions are preferably minimal; typically four, three, two or one amino acid difference relative to the human immunoglobulin sequence or consensus framework sequence.
非人抗体的高变区残基被并入到VL和/或VH受体人框架中。例如，可以并入对应于Kabat CDR残基、Chothia高变环残基、Abm残基和/或接触残基的残基。任选地，并入如下的延长的高变区残基：24-36或24-34(L1)、46-56或50-56(L2)和89-97(L3)，26-35B(H1)，50-65、47-65或49-65(H2)和93-102、94-102或95-102(H3)。The hypervariable region residues of the non-human antibody are incorporated into the VL and/or VH acceptor human framework. For example, residues corresponding to Kabat CDR residues, Chothia hypervariable loop residues, Abm residues, and/or contact residues may be incorporated. Optionally, the following extended hypervariable region residues are incorporated: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3), 26-35B (H1 ), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3).
在一些方面，用于缀合物的抗-因子D抗体或抗体变体包含轻链结构域和重链可变结构域。在一些方面，参比抗-因子D抗体包含SEQ ID NO:3的轻链可变结构域。在一些方面，参比抗-因子D抗体包含SEQ ID NO:4的重链可变结构域。In some aspects, the anti-Factor D antibody or antibody variant used in the conjugate comprises a light chain domain and a heavy chain variable domain. In some aspects, the reference anti-Factor D antibody comprises the light chain variable domain of SEQ ID NO:3. In some aspects, the reference anti-Factor D antibody comprises the heavy chain variable domain of SEQ ID NO:4.
此外，抗-因子D抗体可以包含任何合适的恒定结构域序列，前提是抗体保持结合因子D的能力。例如，在一些实施方案中，用于本发明的缀合物的抗-因子D抗体包含至少部分的重链恒定结构域。在一些实施方案中，抗-因子D抗体包含α、δ、ε、γ或μ重链中的一个或其组合的重链恒定结构域。根据其重链恒定结构域(CH)的氨基酸序列，可以将免疫球蛋白分配为不同的类别或同种型。存在五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其重链分别被指定为α、δ、ε、γ和μ。基于在CH序列和功能上相对小的差异，γ和α类被进一步划分为亚类，例如，人表达以下亚类：IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。在一些实施方案中，用于本发明的缀合物的抗-因子D抗体包含重链恒定结构域，所述重链恒定结构域包含在对效应子功能(例如结合亲和力)产生所需影响的氨基酸位置处的置换。在一些实施方案中，用于本发明的缀合物的抗-因子D抗体包含重链恒定结构域，所述重链恒定结构域包含在对效应子功能(例如结合亲和力)不产生影响的氨基酸位置处的置换。在一些实施方案中，抗-因子D抗体包含IgG型(例如IgG1、IgG2、IgG3或IgG4)的重链恒定结构域并且进一步包含位置114(Kabat编号；相当于EU编号中的118)、168(Kabat编号；相当于EU编号中的172)、172(Kabat编号；相当于EU编号中的176)和/或228(EU编号)处的置换。在一些实施方案中，抗-因子D抗体包含IgG(例如IgG1、IgG2、IgG3或IgG4)型的重链恒定结构域并且进一步包含位置114处的置换，其中位置114是半胱氨酸(C)或丙氨酸(A)，位置168是半胱氨酸(C)或丙氨酸(A)，位置172是半胱氨酸(C)或丙氨酸(A)和/或位置228是脯氨酸(P)、精氨酸(R)或丝氨酸(S)。Furthermore, an anti-Factor D antibody may comprise any suitable constant domain sequence, provided the antibody retains the ability to bind Factor D. For example, in some embodiments, an anti-Factor D antibody used in a conjugate of the invention comprises at least a portion of a heavy chain constant domain. In some embodiments, the anti-Factor D antibody comprises a heavy chain constant domain of one or a combination of alpha, delta, epsilon, gamma, or mu heavy chains. Depending on the amino acid sequence of the constant domain (CH ) of their heavy chains, immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, the heavy chains of which are designated α, δ, ε, γ, and μ, respectively. The gamma and alpha classes are further divided into subclasses based on relatively small differences inCH sequence and function, eg, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgAl and IgA2. In some embodiments, the anti-Factor D antibodies used in the conjugates of the invention comprise a heavy chain constant domain comprising the desired effect on effector function (e.g., binding affinity). Substitutions at amino acid positions. In some embodiments, the anti-Factor D antibodies used in the conjugates of the invention comprise a heavy chain constant domain comprising amino acids that do not affect effector function (e.g., binding affinity). Replacement at position. In some embodiments, the anti-Factor D antibody comprises a heavy chain constant domain of IgG type (e.g., IgG1, IgG2, IgG3, or IgG4) and further comprises positions 114 (Kabat numbering; equivalent to 118 in EU numbering), 168 ( Kabat numbering; equivalent to 172 in EU numbering), 172 (Kabat numbering; equivalent to 176 in EU numbering) and/or substitutions at 228 (EU numbering). In some embodiments, the anti-Factor D antibody comprises a heavy chain constant domain of IgG (e.g., IgGl, IgG2, IgG3, or IgG4) type and further comprises a substitution at position 114, wherein position 114 is cysteine (C) or alanine (A), position 168 is cysteine (C) or alanine (A), position 172 is cysteine (C) or alanine (A) and/or position 228 is pro amino acid (P), arginine (R) or serine (S).
此外，例如，在一些实施方案中，用于本发明的缀合物的抗-因子D抗体包含至少部分的轻链恒定结构域。在一些实施方案中，抗-因子D抗体包含κ或λ轻链中的一个或其组合的轻链恒定结构域，因为来自任何脊椎动物物种的轻链都可以基于其恒定结构域的氨基酸序列而被分配至两个明显不同的类别(称为κ和λ)中的一个。在一些实施方案中，用于本发明的缀合物的抗-因子D抗体包含轻链恒定结构域，所述轻链恒定结构域包含在对效应子功能(例如结合亲和力)产生所需影响的氨基酸位置处的置换。在一些实施方案中，用于本发明的缀合物的抗-因子D抗体包含轻链恒定结构域，所述轻链恒定结构域包含在对效应子功能(例如结合亲和力)不产生影响的氨基酸位置处的置换。在一些实施方案中，用于本发明的缀合物的抗-因子D抗体包含κ型的轻链恒定结构域并且进一步包含位置110、144、146和/或168(Kabat编号)处的置换。在一些实施方案中，用于本发明的缀合物的抗-因子D抗体包含κ型的轻链恒定结构域并且进一步包含位置110、位置144、位置146和/或位置168处的置换，其中110是半胱氨酸(C)或缬氨酸(V)，144是半胱氨酸(C)或丙氨酸(A)，146是异亮氨酸(I)或缬氨酸(V)，168是半胱氨酸(C)或丝氨酸(S)。Also, for example, in some embodiments, an anti-Factor D antibody useful in a conjugate of the invention comprises at least a portion of a light chain constant domain. In some embodiments, an anti-Factor D antibody comprises a light chain constant domain of one or a combination of kappa or lambda light chains, since light chains from any vertebrate species can be identified based on the amino acid sequence of their constant domains. are assigned to one of two distinct classes, called κ and λ. In some embodiments, the anti-Factor D antibodies used in the conjugates of the invention comprise a light chain constant domain comprising the desired effect on effector function (e.g., binding affinity). Substitutions at amino acid positions. In some embodiments, the anti-Factor D antibodies used in the conjugates of the invention comprise a light chain constant domain comprising amino acids that have no effect on effector function (e.g., binding affinity). Replacement at position. In some embodiments, the anti-Factor D antibody used in the conjugates of the invention comprises a light chain constant domain of the kappa type and further comprises substitutions at positions 110, 144, 146 and/or 168 (Kabat numbering). In some embodiments, the anti-Factor D antibody used in the conjugates of the invention comprises a light chain constant domain of the kappa type and further comprises a substitution at position 110, position 144, position 146 and/or position 168, wherein 110 is cysteine (C) or valine (V), 144 is cysteine (C) or alanine (A), 146 is isoleucine (I) or valine (V) , 168 is cysteine (C) or serine (S).
可以修饰包括人源化抗-因子D抗体在内的亲本或参比抗-因子D抗体以产生修饰的抗-因子D抗体或抗-因子D抗体变体。在一些实施方案中，修饰的抗-因子D抗体及其变体相比于亲本抗体可以具有改善的物理、化学、生物学或均一性性质。Parental or reference anti-Factor D antibodies, including humanized anti-Factor D antibodies, can be modified to produce modified anti-Factor D antibodies or anti-Factor D antibody variants. In some embodiments, modified anti-Factor D antibodies and variants thereof may have improved physical, chemical, biological or homogeneity properties compared to the parent antibody.
在一些实施方案中，用于本发明的缀合物的抗体的一个或多个氨基酸改变(例如置换)成亲本抗体高变区的一个或多个氨基酸。备选地或另外地，可以将框架区的一个或多个改变(例如置换)引入亲本抗体中。修饰的框架区残基的实例包括直接与抗原非共价结合(Amit等人,(1986)Science,233:747-753)；与CDR相互作用/影响CDR的构象(Chothia等人(1987)J.Mol.Biol.,196:901-917)和/或参与VL-VH界面(EP 239 400B1)的那些。在某些实施方案中，一个或多个此种框架区残基的修饰导致抗体对抗原的结合亲和力增强。例如，在本发明的该实施方案中，约一个至约5个框架残基可以被改变。修饰的框架或HVR区残基的实例包括这样的位点，其中在所述位点处的修饰导致产生脱酰胺的变体(例如，天冬酰胺(N或Asn)残基被修饰成天冬氨酸(D或Asp)，氧化变体(例如，甲硫氨酸(M或Met)残基和/或色氨酸(W或Trp)残基被修饰成砜或亚砜)或焦谷氨酸变体(例如，谷氨酰胺(Q或Gln)残基被修饰成焦谷氨酸)。修饰的框架区残基或HVR区残基的实例包括可能的脱酰胺位点(即，天冬酰胺(N或Asn))、氧化位点(即，甲硫氨酸(M或Met)或色氨酸(W或Trp))或焦谷氨酸转化位点(即，谷氨酰胺(Q或Gln))，其中这些位点处的修饰分别阻止脱酰胺作用和/或氧化和/或焦谷氨酸转化。In some embodiments, one or more amino acids of the antibody used in the conjugates of the invention are changed (eg, substituted) with one or more amino acids of the hypervariable region of the parent antibody. Alternatively or additionally, one or more alterations (eg substitutions) of the framework regions may be introduced into the parent antibody. Examples of modified framework residues include direct non-covalent binding to antigen (Amit et al., (1986) Science, 233:747-753); interaction with/affecting the conformation of CDRs (Chothia et al. (1987) J Mol. Biol., 196:901-917) and/or those involved in the VL-VH interface (EP 239 400B1). In certain embodiments, modification of one or more such framework region residues results in increased binding affinity of the antibody for the antigen. For example, about one to about five framework residues may be altered in this embodiment of the invention. Examples of modified framework or HVR region residues include sites where modification results in a deamidated variant (e.g., an asparagine (N or Asn) residue modified to asparagine Acids (D or Asp), oxidized variants (e.g., methionine (M or Met) residues and/or tryptophan (W or Trp) residues modified to sulfones or sulfoxides), or pyroglutamate Variants (e.g., glutamine (Q or Gln) residues modified to pyroglutamic acid). Examples of modified framework region residues or HVR region residues include possible deamidation sites (i.e., asparagine (N or Asn)), oxidation sites (i.e., methionine (M or Met) or tryptophan (W or Trp)), or pyroglutamate conversion sites (i.e., glutamine (Q or Gln )), wherein modifications at these sites prevent deamidation and/or oxidation and/or pyroglutamic acid conversion, respectively.
为了防止形成脱酰胺变体，可以将天冬酰胺(N或Asn)突变成丙氨酸(A或Ala)、谷氨酰胺(Q或Gln)或丝氨酸(S或Ser)。为了防止形成氧化变体，可以将甲硫氨酸(Met)或色氨酸(W或Trp)突变成亮氨酸(L)或异亮氨酸(I)。为了防止形成焦谷氨酸变体，可以将谷氨酰胺(Q或Gln)突变成谷氨酸(E或Glu)。(Amphlett,G.等人,Pharm.Biotechnol.,9:1-140(1996))。备选地或另外地，框架区残基的一个或多个改变(例如置换)可以在亲本抗体的Fc区中。To prevent the formation of deamidated variants, asparagine (N or Asn) can be mutated to alanine (A or Ala), glutamine (Q or Gln) or serine (S or Ser). To prevent the formation of oxidative variants, methionine (Met) or tryptophan (W or Trp) can be mutated to leucine (L) or isoleucine (I). To prevent the formation of pyroglutamate variants, glutamine (Q or Gln) can be mutated to glutamic acid (E or Glu). (Amphlett, G. et al., Pharm. Biotechnol., 9:1-140 (1996)). Alternatively or additionally, one or more alterations (eg substitutions) of framework region residues may be in the Fc region of the parent antibody.
一种可用于产生此种修饰抗体的方法被称为“丙氨酸扫描诱变”(Cunningham和Wells(1989)Science 244:1081-1085)。这里，一个或多个高变区残基被丙氨酸或聚丙氨酸残基代替从而影响氨基酸与抗原的相互作用。然后，通过在置换位点处或者针对置换位点引入另外的或其它突变来精修对置换显示功能敏感性的那些高变区残基。因此，虽然用于引入氨基酸序列变化的位点是预先确定的，但是突变本身的性质不需要预先确定。筛选以此方式产生的ala突变体的生物学活性(即结合亲和力或溶血测定)，如本文所述。One method that can be used to generate such modified antibodies is called "alanine scanning mutagenesis" (Cunningham and Wells (1989) Science 244:1081-1085). Here, one or more hypervariable region residues are replaced by alanine or polyalanine residues to affect the interaction of the amino acids with the antigen. Those hypervariable region residues showing functional sensitivity to the substitution are then refined by introducing additional or other mutations at or for the substitution site. Thus, while the sites for introducing amino acid sequence changes are predetermined, the nature of the mutation itself need not be predetermined. The ala mutants generated in this manner are screened for biological activity (ie, binding affinity or hemolysis assays), as described herein.
即使在抗体或其片段(例如抗原结合片段)中有更多的修饰，也可以通过选择对保持以下方面的影响显著不同的置换来实现生物学性质：(a)置换区域中多肽主链的结构，例如，呈折叠或螺旋构型，(b)分子在靶位点处的电荷或疏水性，或(c)侧链的大小。基于共有的侧链性质，天然存在的残基被划分成多组：Even with more modifications in an antibody or fragment thereof (such as an antigen-binding fragment), biological properties can be achieved by selecting substitutions that have significantly different effects on maintaining (a) the structure of the polypeptide backbone in the region of the substitution , for example, in a folded or helical configuration, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Naturally occurring residues are divided into groups based on shared side chain properties:
(1)疏水：正亮氨酸、met、ala、val、leu、ile；(1) Hydrophobic: norleucine, met, ala, val, leu, ile;
(2)中性亲水：cys、ser、thr、asn、gln；(2) Neutral hydrophilic: cys, ser, thr, asn, gln;
(3)酸性：asp、glu；(3) Acidity: asp, glu;
(4)碱性：his、lys、arg；(4) Basic: his, lys, arg;
(5)影响链取向的残基：gly、pro；和(5) Residues affecting chain orientation: gly, pro; and
(6)芳香性：trp、tyr、phe。(6) Aromaticity: trp, tyr, phe.
非保守性置换将需要将这些类别之一的成员交换为另一个类别的成员。Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.
在另一个实施方案中，修饰被选择用于修饰的位点，并且通过噬菌体展示选择具有提高的结合亲和力的那些修饰。In another embodiment, modifications are selected for the site of modification, and those modifications with improved binding affinity are selected by phage display.
通过本领域已知的多种方法来制备编码氨基酸序列突变体或修饰的氨基酸序列的核酸分子。这些方法包括但不限于对早期制备的变体或非变体形式的亲本抗体进行寡核苷酸介导的(或定点)诱变、PCR诱变和盒式诱变。一种用于制备突变体或变体或修饰的氨基酸序列的方法是定点诱变(参见，例如，Kunkel(1985)Proc.Natl.Acad.Sci.USA 82:488)。Nucleic acid molecules encoding amino acid sequence mutants or modified amino acid sequences are prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of variant or non-variant forms of the parent antibody prepared earlier. One method for making mutants or variants or modified amino acid sequences is site-directed mutagenesis (see, eg, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488).
在某些实施方案中，修饰的抗体将仅具有单个置换的高变区残基。在其它实施方案中，亲本抗体的高变区残基中的两个或更多个将被置换，例如约两个至约十个高变区置换。通常，修饰的抗体的氨基酸序列与亲本抗体的重链可变结构域或轻链可变结构域的氨基酸序列具有至少75％，更优选至少80％，更优选至少85％，更优选至少90％，并且最优选至少95％的氨基酸序列同一性或相似性(以上在定义部分被定义)。In certain embodiments, a modified antibody will have only a single hypervariable region residue substituted. In other embodiments, two or more of the hypervariable region residues of the parent antibody will be substituted, eg, about two to about ten hypervariable region substitutions. Typically, the amino acid sequence of the modified antibody is at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% identical to the amino acid sequence of the heavy chain variable domain or the light chain variable domain of the parental antibody. , and most preferably at least 95% amino acid sequence identity or similarity (defined above in the definitions section).
在制备修饰的抗体后，确定所述分子相对于亲本抗体的生物学活性。如上所述，这可以包括确定抗体变体或其片段(例如抗原结合片段)的结合亲和力和/或其它生物学活性。在本发明的一些实施方案中，制备一系列修饰的抗体并筛选其对抗原如因子D或其片段的结合亲和力。任选地对选自该初筛的一个或多个抗体突变体或修饰的抗体进行一种或多种进一步的生物学活性测定以确认所述抗体变体或其片段(例如抗原结合片段)确实可用于例如临床前研究。After making the modified antibody, the biological activity of the molecule is determined relative to the parent antibody. As noted above, this may include determining the binding affinity and/or other biological activity of the antibody variant or fragment thereof (eg, an antigen-binding fragment). In some embodiments of the invention, a series of modified antibodies are prepared and screened for binding affinity to an antigen, such as Factor D or a fragment thereof. One or more further biological activity assays are optionally performed on one or more antibody mutants or modified antibodies selected from this primary screen to confirm that the antibody variants or fragments thereof (e.g., antigen-binding fragments) do Can be used eg in preclinical research.
可以对本文所述的修饰的抗-因子D抗体进行进一步修饰，通常这取决于修饰的抗体的目的用途。所述修饰可以包括氨基酸序列的进一步改变，与异源多肽融合和/或共价修饰如以下详述的那些。关于氨基酸序列改变，以上详述了示例性修饰。例如，也可以置换任何不涉及保持修饰的抗体的正确构象的半胱氨酸残基(通常用丝氨酸置换)，以改善分子的氧化稳定性和防止异常交联。相反地，可以向抗体中添加半胱氨酸键以改善其稳定性(特别是当抗体是抗体片段诸如Fv片段时)。Further modifications may be made to the modified anti-Factor D antibodies described herein, generally depending on the intended use of the modified antibody. Such modifications may include further changes in amino acid sequence, fusions to heterologous polypeptides and/or covalent modifications such as those detailed below. With respect to amino acid sequence changes, exemplary modifications are detailed above. For example, any cysteine residue not involved in maintaining the correct conformation of the modified antibody may also be substituted (usually with serine) to improve the oxidative stability of the molecule and to prevent aberrant crosslinking. Conversely, cysteine linkages can be added to antibodies to improve their stability (especially when the antibody is an antibody fragment such as an Fv fragment).
另一类氨基酸突变体具有改变的糖基化模式。这可以通过以下实现：删除存在于抗体中的一个或多个碳水化合物部分，和/或添加抗体中不存在的一个或多个糖基化位点。抗体或抗体片段(例如抗原结合片段)的糖基化通常是N-连接的或O-连接的。N-连接是指碳水化合物部分连接于天冬酰胺残基的侧链。三肽序列天冬酰胺-X-丝氨酸和天冬酰胺-X-苏氨酸(其中X是除脯氨酸以外的任何氨基酸)是用于将碳水化合物部分酶促连接于天冬酰胺侧链的识别序列。因此，在多肽中存在这些三肽序列中的任一个产生了潜在的糖基化位点。O-连接的糖基化是指将糖N-乙酰半乳糖胺、半乳糖或木糖之一连接于羟基氨基酸，最常见的是丝氨酸或苏氨酸，但是也可以使用5-羟脯氨酸或5-羟赖氨酸。向抗体中添加糖基化位点可以通过改变氨基酸序列使得其含有一个或多个上述三肽序列而便利地完成(用于N-连接的糖基化位点)。还可以通过向原始抗体的序列中添加或置换一个或多个丝氨酸或苏氨酸残基来进行所述改变(用于O-连接的糖基化位点)。Another class of amino acid mutants has altered glycosylation patterns. This can be achieved by deleting one or more carbohydrate moieties present in the antibody, and/or adding one or more glycosylation sites not present in the antibody. Glycosylation of antibodies or antibody fragments (eg, antigen-binding fragments) is typically N-linked or O-linked. N-linked means that the carbohydrate moiety is attached to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are used to enzymatically attach the carbohydrate moiety to the asparagine side chain recognition sequence. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline can also be used or 5-hydroxylysine. Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The alteration may also be made by the addition or substitution of one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
抗-因子D抗体及其变体的亲和力和生物活性Affinity and biological activity of anti-factor D antibodies and variants thereof
针对理想性质如因子D-结合亲和力和因子D-抑制活性，可以在体外或体内筛选具有在本文中被鉴定为对于抗-因子D抗体来说理想的特性的抗体。Antibodies with the properties identified herein as desirable for anti-Factor D antibodies can be screened for desirable properties such as Factor D-binding affinity and Factor D-inhibitory activity in vitro or in vivo.
a.亲和力a. Affinity
在一些方面，用于本发明的缀合物的抗-因子D抗体变体与其所来源于的亲本抗-因子D抗体竞争。还提供结合与亲本抗-因子D抗体相同的表位的抗-因子D抗体变体。In some aspects, the anti-Factor D antibody variant used in the conjugates of the invention competes with the parent anti-Factor D antibody from which it was derived. Anti-Factor D antibody variants that bind to the same epitope as the parental anti-Factor D antibody are also provided.
为了确定抗-因子D抗体变体是否结合人因子D上的参比抗-因子D抗体所结合的相同表位，可以进行交叉阻断测定(Antibodies,ALaboratory Manual,Cold Spring HarborLaboratory,Ed Harlow and David Lane(1988))。备选地，可以进行表位定位(mapping)以确定抗-因子D抗体是否结合目标表位(Champe等人(1995)J.Biol.Chem.270:1388-1394。抗体对例如人因子D的亲和力可以使用标准方法确定，所述方法包括实施例中更为详细描述的表面等离子共振(SPR)测定。To determine whether an anti-Factor D antibody variant binds the same epitope on human Factor D as a reference anti-Factor D antibody, a cross-blocking assay can be performed (Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)). Alternatively, epitope mapping can be performed to determine whether an anti-Factor D antibody binds an epitope of interest (Champe et al. (1995) J. Biol. Chem. 270: 1388-1394. Antibody against, for example, human Factor D Affinity can be determined using standard methods including the Surface Plasmon Resonance (SPR) assay described in more detail in the Examples.
在一些方面，用于本发明的缀合物的抗-因子D抗体变体的因子D结合亲和力与其所来源于的亲本抗-因子D抗体的因子D结合亲和力相当。在一些方面，用于本发明的缀合物的抗-因子D抗体变体的因子D结合亲和力在亲本抗-因子D抗体的因子D结合亲和力的10倍、7倍、5倍、2倍或1倍内。In some aspects, the Factor D binding affinity of the anti-Factor D antibody variant used in the conjugates of the invention is comparable to the Factor D binding affinity of the parent anti-Factor D antibody from which it is derived. In some aspects, the Factor D binding affinity of the anti-Factor D antibody variant used in the conjugates of the invention is 10 times, 7 times, 5 times, 2 times, or 2 times the Factor D binding affinity of the parental anti-Factor D antibody. Within 1 times.
在一些实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为20nM(20x10-9M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为10nM(10x10-9M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为1.0nM(1.0x10-9M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为0.5nM(0.5x10-9M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为1.0pM(1.0x10-12M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为0.5pM(0.5x10-12M)或更好。In some embodiments, the invention provides conjugates comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is 20 nM (20×10−9 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the monovalent form of the antibody has an affinity for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is 10 nM (10×10− 9M ) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the monovalent form of the antibody has an affinity for Factor D (e.g., an antibody as a Fab fragment has an affinity for Factor D) of 1.0 nM (1.0 nM). x10-9 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the monovalent form of the antibody has an affinity for Factor D (e.g., an antibody as a Fab fragment has an affinity for Factor D) of 0.5 nM (0.5 x10-9 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is 1.0 pM (1.0x10-12 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is 0.5 pM (0.5x10-12 M) or better.
在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为10.0nM(10.0x10-9M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为5.0nM(5.0x10-9M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为1.0nM(1.0x10-9M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为0.5nM(0.5x10-9M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为5.0pM(5.0x10-12M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为2.0pM(2.0x10-12M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为1.0pM(1.0x10-12M)或更好。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为0.5pM(0.5x10-12M)或更好。In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the affinity of the antibody for Factor D as an IgG) of 10.0 nM (10.0 x10-9 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the affinity of the antibody for Factor D as an IgG) of 5.0 nM (5.0 nM). x10-9 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the antibody has an affinity for Factor D as an IgG) of 1.0 nM (1.0 nM). x10-9 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the antibody has an affinity for Factor D as an IgG) of 0.5 nM (0.5 x10-9 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the affinity of the antibody for Factor D as an IgG) of 5.0 pM (5.0 pM).x10-12 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the affinity of the antibody for Factor D as an IgG) of 2.0 pM (2.0 pM).x10-12 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the affinity of the antibody for Factor D as an IgG) of 1.0 pM (1.0 pM).x10-12 M) or better. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the affinity of the antibody for Factor D as an IgG) of 0.5 pM (0.5x10-12 M) or better.
在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为0.5mM(0.5x10-6M)至0.5pM(0.5x10-12M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为15nM(15x10-9M)至0.1nM(0.1x10-9M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为5.5nM(5.5x10-9M)至1nM(1x10-9M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为0.5pM(0.5x10-12M)至50pM(5x10-11M)。In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (eg, the affinity of the antibody as a Fab fragment for Factor D) is 0.5 mM (0.5 x10-6 M) to 0.5 pM (0.5x10-12 M). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the monovalent form of the antibody has an affinity for Factor D (e.g., an antibody as a Fab fragment has an affinity for Factor D) of 15 nM (15×10− 9 M) to 0.1 nM (0.1x10-9 M). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (eg, the affinity of the antibody as a Fab fragment for Factor D) is 5.5 nM (5.5 nM). x10-9 M) to 1 nM (1x10-9 M). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is 0.5 pM (0.5 x10-12 M) to 50 pM (5x10-11 M).
在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为0.5mM(0.5x10-6M)至0.5pM(0.5x10-12M)。在另一个实施方案中，本发明提供包含抗-因子D抗体或其抗体变体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为10nM(10x10-9M)至0.05nM(0.05x10-9M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为5.5nM(5.5x10-9M)至1nM(1x10-9M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为0.5pM(0.5x10-12M)至50pM(5x10-11M)。In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the antibody has an affinity for Factor D as an IgG) of 0.5 mM (0.5 x10-6 M) to 0.5 pM (0.5x10-12 M). In another embodiment, the invention provides conjugates comprising an anti-Factor D antibody or antibody variant thereof, wherein the affinity of the antibody for Factor D in a bivalent form (e.g., the affinity of the antibody for Factor D as an IgG) From 10nM (10x10-9 M) to 0.05nM (0.05x10-9 M). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the affinity of the antibody for Factor D as an IgG) of 5.5 nM (5.5 nM). x10-9 M) to 1 nM (1x10-9 M). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the affinity of the antibody for Factor D as an IgG) of 0.5 pM (0.5 x10-12 M) to 50 pM (5x10-11 M).
在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为约1.4pM(1.4x10-12M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为约1.1pM(1.1x10-12M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为约0.19nM(0.19x10-9M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为约0.08nM(0.08x10-9M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为约12.3nM(12.3x10-9M)。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为约9.0nM(9.0x10-9M)。In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is about 1.4 pM ( 1.4x10-12M ). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the antibody has an affinity for Factor D as an IgG) of about 1.1 pM (1.1x10-12M ). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is about 0.19 nM ( 0.19x10-9 M). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the antibody has an affinity for Factor D as an IgG) of about 0.08 nM ( 0.08x10-9 M). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is about 12.3 nM (12.3x10-9 M). In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the antibody has an affinity for Factor D as an IgG) of about 9.0 nM ( 9.0x10-9 M).
在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为约1.4pM(1.4x10-12M)+/-0.5。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为约1.1pM(1.1x10-12M)+/-0.6。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为约0.19nM(0.19x10-9M)+/-.01。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为约0.08nM(0.08x10-9M)+/-0.01。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中单价形式的抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)为约12.3nM(12.3x10-9M)+/-2。在另一个实施方案中，本发明提供包含抗-因子D抗体的缀合物，其中二价形式的抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)为约9.0nM(9.0x10-9M)+/-1。In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is about 1.4 pM ( 1.4x10-12 M) +/-0.5. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the antibody has an affinity for Factor D as an IgG) of about 1.1 pM ( 1.1x10-12 M) +/-0.6. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is about 0.19 nM ( 0.19x10-9 M) +/-.01. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the antibody has an affinity for Factor D as an IgG) of about 0.08 nM ( 0.08x10-9 M) +/-0.01. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the affinity of the monovalent form of the antibody for Factor D (e.g., the affinity of the antibody as a Fab fragment for Factor D) is about 12.3 nM ( 12.3x10-9 M) +/-2. In another embodiment, the invention provides a conjugate comprising an anti-Factor D antibody, wherein the bivalent form of the antibody has an affinity for Factor D (e.g., the antibody has an affinity for Factor D as an IgG) of about 9.0 nM ( 9.0x10-9 M) +/-1.
在另一个实施方案中，单价形式的用于本发明的缀合物的抗-因子D抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)可以为约1.4pM(1.4x10-12M)+/-2。在另一个实施方案中，二价形式的用于本发明的缀合物的抗-因子D抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)可以为约1.1pM(1.1x10-12M)+/-2。在另一个实施方案中，单价形式的用于本发明的缀合物的抗-因子D抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)可以为约0.19nM(0.19x10-9M)+/-2。在另一个实施方案中，二价形式的用于本发明的缀合物的抗-因子D抗体或其抗体变体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)可以为约0.08nM(0.08x10-9M)+/-2。在另一个实施方案中，单价形式的用于本发明的缀合物的抗-因子D抗体对因子D的亲和力(例如，作为Fab片段的抗体对因子D的亲和力)可以为约12.3nM(12.3x10-9M)+/-2。在另一个实施方案中，二价形式的用于本发明的缀合物的抗-因子D抗体对因子D的亲和力(例如，作为IgG的抗体对因子D的亲和力)可以为约9.0nM(9.0x10-9M)+/-2。In another embodiment, the affinity for Factor D of the anti-Factor D antibody used in the conjugates of the invention in monovalent form (e.g., the affinity of the antibody as a Fab fragment for Factor D) may be about 1.4 pM (1.4 x10-12 M) +/-2. In another embodiment, the bivalent form of the anti-Factor D antibody used in the conjugates of the invention may have an affinity for Factor D (e.g., the affinity of the antibody for Factor D as an IgG) of about 1.1 pM (1.1 pM). x10-12 M) +/-2. In another embodiment, the monovalent form of the anti-Factor D antibody used in the conjugates of the invention may have an affinity for Factor D (eg, the affinity of the antibody as a Fab fragment for Factor D) of about 0.19 nM (0.19 nM). x10-9 M) +/-2. In another embodiment, the affinity for Factor D of the bivalent form of the anti-Factor D antibody or antibody variant thereof used in the conjugates of the invention (e.g., the affinity of the antibody as an IgG for Factor D) may be About 0.08nM (0.08x10-9 M) +/-2. In another embodiment, the affinity for Factor D of the anti-Factor D antibody used in the conjugates of the invention in monovalent form (e.g., the affinity of the antibody as a Fab fragment for Factor D) may be about 12.3 nM (12.3 x10-9 M) +/-2. In another embodiment, the bivalent form of the anti-Factor D antibody used in the conjugates of the invention may have an affinity for Factor D (e.g., an antibody that is an IgG that has an affinity for Factor D) of about 9.0 nM (9.0 nM). x10-9 M) +/-2.
如本领域中已知的，配体对其受体的结合亲和力可以使用多种测定中的任一种来确定，并且可以表示为多种数量值。因此，在一些实施方案中，结合亲和力以KD值表示并且反映固有结合亲和力(例如，在亲合力作用最小的情况下)。通常并且优选地，在体外(在无细胞情况下或在细胞相关情况下)测量结合亲和力。如本文中更为详细地描述的，结合亲和力的倍数差异可以被量化为人源化抗体(例如，Fab形式的)的单价结合亲和力值与参比/比较抗体(例如，Fab形式的)(例如，具有供体高变区序列的鼠抗体)的单价结合亲和力值之比，其中在相似的测定条件下测定结合亲和力值。因此，在一些实施方案中，结合亲和力的倍数差异被确定为Fab形式的人源化抗体与所述参比/比较Fab抗体的KD值之比。例如，在一些实施方案中，如果本发明的抗体(A)的亲和力比参比抗体(M)的亲和力“3倍低”，那么如果A的KD值是3x，则M的KD值将是1x，并且A的KD与M的KD之比将是3:1。相反，在一些实施方案中，如果本发明的抗体(C)的亲和力比参比抗体(R)的亲和力“3倍高”，那么如果C的KD值是1x，则R的KD值将是3x，并且C的KD与R的KD之比将是1:3。包括本文中所述的那些在内的本领域中已知的多种测定中的任一种可以用于获得结合亲和力量度，所述测定包括例如Biacore、放射性免疫测定(RIA)和ELISA。As is known in the art, the binding affinity of a ligand for its receptor can be determined using any of a variety of assays and can be expressed as a variety of quantitative values. Thus, in some embodiments, binding affinity is expressed as aKD value and reflects intrinsic binding affinity (eg, where the effect of avidity is minimal). Typically, and preferably, binding affinity is measured in vitro (either in a cell-free setting or in a cell-associated setting). As described in more detail herein, the fold difference in binding affinity can be quantified as the monovalent binding affinity value of a humanized antibody (e.g., in Fab format) compared to a reference/comparator antibody (e.g., in Fab format) (e.g., Ratio of monovalent binding affinity values for a murine antibody having a donor hypervariable region sequence), wherein the binding affinity values were determined under similar assay conditions. Thus, in some embodiments, the fold difference in binding affinity is determined as the ratio of theKD values of the humanized antibody in Fab form to said reference/comparative Fab antibody. For example, in some embodiments, if the affinity of an antibody of the invention (A) is "3-fold lower" than the affinity of a reference antibody (M), then if theK value of A is 3x, theK value of M will be is 1x, and the ratio of A's KD to M's KD will be 3:1. Conversely, in some embodiments, if the affinity of an antibody of the invention (C) is "3-fold higher" than that of a reference antibody (R), then if theK value of C is 1x, theK value of R will be is 3x, and the ratio of C'sKD to R'sKD will be 1:3. Any of a variety of assays known in the art, including those described herein, can be used to obtain a measure of binding affinity, including, for example, Biacore, radioimmunoassay (RIA), and ELISA.
此外，用于本发明的缀合物的抗体的KD值可以变化，这取决于使用的具体测定的条件。例如，在一些实施方案中，结合亲和力量度可以在这样的测定中获得，其中将Fab或抗体固定并且测量配体(即因子D)的结合，或备选地，将Fab或抗体的配体(即因子D)固定并且测量Fab或抗体的结合。在一些实施方案中，结合亲和力量度可以在这样的测定中获得，其中再生条件可以包含(1)10mM甘氨酸或4M MgCl2，pH 1.5，以及(2)pH 1.0至pH 7.5之间的pH，包括pH 1.5、pH 5.0、pH 6.0和pH 7.2。在一些实施方案中，结合亲和力量度可以在这样的测定中获得，其中结合条件可以包含(1)PBS或HEPES-缓冲盐水和(2)Tween-20，即0.1％Tween-20。在一些实施方案中，结合亲和力量度可以在这样的测定中获得，其中配体(即因子D)的来源可以来自可商购的来源。在一些实施方案中，结合亲和力量度可以在这样的测定中获得，其中(1)将Fab或抗体固定并且测量配体(即因子D)的结合，(2)再生条件包含4MMgCl2，pH 7.2，并且(3)结合条件包含HEPES-缓冲盐水，pH 7.2，含0.1％Tween-20。在一些实施方案中，结合亲和力量度可以在这样的测定中获得，其中(1)将配体(即因子D)固定并测量Fab或抗体的结合，(2)再生条件包含10mM甘氨酸，pH 1.5，并且(3)结合条件包含PBS缓冲剂。Furthermore, theKD values of the antibodies used in the conjugates of the invention can vary depending on the conditions of the particular assay used. For example, in some embodiments, binding affinity measures can be obtained in assays in which the Fab or antibody is immobilized and the binding of the ligand (i.e., Factor D) is measured, or alternatively, the ligand of the Fab or antibody ( That is, Factor D) is immobilized and the binding of the Fab or antibody is measured. In some embodiments, binding affinity measures can be obtained in assays wherein regeneration conditions can comprise (1) 10 mM glycine or 4M MgCl2 , pH 1.5, and (2) pH between pH 1.0 and pH 7.5, including pH 1.5, pH 5.0, pH 6.0 and pH 7.2. In some embodiments, a measure of binding affinity can be obtained in an assay wherein binding conditions can comprise (1) PBS or HEPES-buffered saline and (2) Tween-20, ie 0.1% Tween-20. In some embodiments, a measure of binding affinity can be obtained in an assay in which the source of the ligand (ie Factor D) can be from a commercially available source. In some embodiments, binding affinity measures can be obtained in assays in which (1) the Fab or antibody is immobilized and binding of the ligand (i.e., Factor D) is measured, (2) regeneration conditions comprise4MMgCl2 , pH 7.2, And (3) binding conditions comprised HEPES-buffered saline, pH 7.2, containing 0.1% Tween-20. In some embodiments, binding affinity measures can be obtained in assays in which (1) the ligand (i.e., Factor D) is immobilized and Fab or antibody binding is measured, (2) regeneration conditions comprise 10 mM glycine, pH 1.5, And (3) the binding condition comprises PBS buffer.
b.生物活性b. Biological activity
为了确定抗因子D抗体或其变体或片段(例如抗原结合片段)是否能够与因子D结合并发挥生物效应(例如抑制旁路途径溶血)，可以使用利用兔RBC进行的溶血抑制测定，包括实施例2中所述的那些。这种溶血抑制可以使用标准测定进行确定(Kostavasili等人(1997)J of Immunology158:1763-72；Wiesmann等人(2006)Nature 444:159-60)。这种测定中的补体激活可以用血清或血浆引发。因子D在血清或血浆中的适合浓度(Pascual等人(1998)Kidney International 34:529-536；Complement Facts Book,Bernard J.Morley和Mark J.Walport,编辑,Academic Press(2000)；Barnum等人(1984)J.Immunol.Methods,67:303-309)可以根据本领域已知的方法常规地确定，包括已经在参考文献中描述的那些，诸如Pascual等人(1998)Kidney International 34:529-536和Barnum等人(1984)J.Immunol.Methods 67:303-309。本发明通常涉及能够抑制与因子D有关的生物活性的抗体。例如，在18μg/ml的浓度下(相当于血液中人因子D的摩尔浓度的约1.5倍；抗-因子D抗体与因子D的摩尔比为约1.5:1)，可以观察到抗体对旁路补体活性的显著抑制(参见例如美国专利号6,956,107)。To determine whether anti-Factor D antibodies or variants or fragments thereof (e.g., antigen-binding fragments) are capable of binding Factor D and exerting a biological effect (e.g., inhibiting alternative pathway hemolysis), a hemolysis inhibition assay using rabbit RBCs can be used, including performing Those described in Example 2. This inhibition of hemolysis can be determined using standard assays (Kostavasili et al. (1997) J of Immunology 158:1763-72; Wiesmann et al. (2006) Nature 444:159-60). Complement activation in this assay can be initiated with serum or plasma. Suitable concentrations of Factor D in serum or plasma (Pascual et al. (1998) Kidney International 34:529-536; Complement Facts Book, Bernard J. Morley and Mark J. Walport, eds., Academic Press (2000); Barnum et al. (1984) J. Immunol. Methods, 67:303-309) can be routinely determined according to methods known in the art, including those already described in references, such as Pascual et al. (1998) Kidney International 34:529- 536 and Barnum et al. (1984) J. Immunol. Methods 67:303-309. The present invention generally relates to antibodies capable of inhibiting biological activities associated with Factor D. For example, at a concentration of 18 μg/ml (corresponding to about 1.5 times the molar concentration of human Factor D in blood; molar ratio of anti-Factor D antibody to Factor D is about 1.5:1), antibody to shunt can be observed Significant inhibition of complement activity (see eg US Pat. No. 6,956,107).
在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值小于30nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值小于15nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值小于10nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值小于5nM。In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of less than 30 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of less than 15 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of less than 10 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with an IC50 value of less than5 nM.
在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为30nM至2nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为25nM至7nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为20nM至12nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为30nM至15nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为12nM至8nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为7nM至2nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为6nM至3nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为8nM至5nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为5nM至2nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为10nM至5nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC550值为8nM至2nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为7nM至3nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为6nM至4nM。在另一个实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为约4.7nM±0.6nM。在另一个实施方案中，本发明涉及抗-因子D抗体，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为约6.4nM±0.6nM。在另一个实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为约3.5nM±0.5nM。在另一个实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为约4.4nM±1.5nM。在另一个实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为约10.2nM±0.8nM。在另一个实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值为约23.9nM±5.0nM。In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 30 nM to 2 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 25 nM to 7 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 20 nM to 12 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 30 nM to 15 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 12 nM to 8 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 7 nM to 2 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 6 nM to 3 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with an IC50 value of 8nM to 5 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with an IC50 value of5 nM to 2 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 10 nM to 5 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC550 value of 8 nM to 2 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 7 nM to 3 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of 6 nM to 4 nM. In another embodiment, the present invention is directed to a conjugate comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of about 4.7 nM ± 0.6 nM. In another embodiment, the invention relates to an anti-Factor D antibody, wherein a Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of about 6.4 nM ± 0.6 nM. In another embodiment, the present invention is directed to a conjugate comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of about 3.5 nM ± 0.5 nM. In another embodiment, the present invention is directed to a conjugate comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of about 4.4 nM ± 1.5 nM. In another embodiment, the present invention is directed to a conjugate comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of about 10.2 nM ± 0.8 nM. In another embodiment, the present invention is directed to a conjugate comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of about 23.9 nM ± 5.0 nM.
在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值小于80nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值小于50nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值小于40nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值小于20nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC50值小于15nM。In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of less than 80 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of less than 50 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of less than 40 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of less than 20 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC50 value of less than 15 nM.
在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为80nM至10nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为75nM至15nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为70nM至20nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为65nM至25nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为60nM至30nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为55nM至35nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为50nM至40nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为80nM至70nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为55nM至25nM。在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为16nM至12nM。在另一个实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为约14.0nM±1.0nM。在另一个实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为约38.0nM±11.0nM。在另一个实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段抑制旁路途径溶血，其中IC90值为约72.6nM±4.8nM。In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 80 nM to 10 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 75 nM to 15 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 70 nM to 20 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 65 nM to 25 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 60 nM to 30 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 55 nM to 35 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 50 nM to 40 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 80 nM to 70 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 55 nM to 25 nM. In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of 16 nM to 12 nM. In another embodiment, the present invention is directed to a conjugate comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of about 14.0 nM±1.0 nM. In another embodiment, the present invention is directed to a conjugate comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of about 38.0 nM±11.0 nM. In another embodiment, the present invention is directed to a conjugate comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis with anIC90 value of about 72.6nM±4.8nM.
在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的Fab片段以如下抗体与因子D摩尔比抑制旁路途径溶血：约0.05:1(0.05)至约10:1(10)、或约0.09:1(0.09)至约8:1(8)、或约0.1:1(0.1)至约6:1(6)、或约0.15:1(0.15)至约5:1(5)、或约0.19:1(0.19)至约4:1(4)、或约0.2:1(0.2)至约3:1(3)、或约0.3:1(0.3)至约2:1(2)、或约0.4:1(0.4)至约1:1(1)、或约0.5:1(0.5)至约1:2(0.5)、或约0.6:1(0.6)至约1:3(0.33)、或约0.7:1(0.7)至约1:4(0.25)、或约0.8:1(0.8)至约1:5(0.2)、或约0.9:1(0.9)至约1:6(0.17)。In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the Fab fragment of said antibody inhibits alternative pathway hemolysis at an antibody to Factor D molar ratio of about 0.05:1 (0.05) to about 10:1(10), or about 0.09:1(0.09) to about 8:1(8), or about 0.1:1(0.1) to about 6:1(6), or about 0.15:1(0.15) to About 5:1 (5), or about 0.19:1 (0.19) to about 4:1 (4), or about 0.2:1 (0.2) to about 3:1 (3), or about 0.3:1 (0.3) to about 2:1(2), or about 0.4:1(0.4) to about 1:1(1), or about 0.5:1(0.5) to about 1:2(0.5), or about 0.6:1(0.6 ) to about 1:3 (0.33), or about 0.7:1 (0.7) to about 1:4 (0.25), or about 0.8:1 (0.8) to about 1:5 (0.2), or about 0.9:1 ( 0.9) to about 1:6 (0.17).
在一些实施方案中，本发明涉及包含人源化抗-因子D抗体的片段(例如抗原结合片段)的缀合物。本发明的抗体片段可以是例如Fv、Fab、Fab-SH、Fab’-SH、Fab’、Fab-C、Fab’-C、Fab’-C-SH、Fab-C-SH、scFv、双抗体、或F(ab’)2、dAb、互补决定区(CDR)片段、线性抗体、单链抗体分子、微抗体(minibody)、双抗体、或由抗体片段形成的多特异性抗体。在另外的实施方案中，本发明涉及包含能够基本上穿透全部视网膜的人源化抗-因子D抗体片段(例如抗原结合片段)的缀合物。在甚至另外的实施方案中，本发明涉及包含能够穿透贯穿整个视网膜厚度的人源化抗-因子D抗体片段(例如抗原结合片段)的缀合物。In some embodiments, the invention relates to conjugates comprising a fragment (eg, an antigen-binding fragment) of a humanized anti-Factor D antibody. Antibody fragments of the invention can be, for example, Fv, Fab, Fab-SH, Fab'-SH, Fab', Fab-C, Fab'-C, Fab'-C-SH, Fab-C-SH, scFv, diabodies , or F(ab')2 , dAb, complementarity determining region (CDR) fragment, linear antibody, single chain antibody molecule, minibody, diabody, or multispecific antibody formed from antibody fragments. In additional embodiments, the invention is directed to conjugates comprising a humanized anti-Factor D antibody fragment (eg, an antigen-binding fragment) capable of penetrating substantially all of the retina. In even further embodiments, the invention is directed to conjugates comprising a humanized anti-Factor D antibody fragment (eg, an antigen-binding fragment) capable of penetrating through the entire thickness of the retina.
在一些实施方案中，本发明涉及包含抗-因子D抗体的缀合物，其中所述抗体的未缀合的Fab片段在经由单次玻璃体内注射施用至哺乳动物眼睛(例如人)后具有至少3、5、7、10或12天的半衰期。在另一个实施方案中，本发明涉及包含人源化抗-因子D抗体的缀合物，其中所述抗体的未缀合的Fab片段在经由单次玻璃体内注射施用至哺乳动物眼睛(例如人)后抑制旁路途径(AP)补体激活达至少40、45、50、55、60、65、70、75、80、85、90、95、100、105、110或115天。在另一个实施方案中，本发明涉及包含人源化抗-因子D抗体的缀合物，其中所述抗体的未缀合的Fab片段抑制旁路途径(AP)补体激活的浓度在经由单次玻璃体内注射施用至哺乳动物眼睛(例如人)后在视网膜组织中维持至少40、45、50、55、60、65、70、75、80或85天。在另一个实施方案中，本发明涉及包含人源化抗-因子D抗体的缀合物，其中所述抗体的未缀合的Fab片段抑制旁路途径(AP)补体激活的浓度在经由单次玻璃体内注射施用至哺乳动物眼睛(例如人)后在玻璃体液中维持至少80、85、90、95、100、105、110或115天。In some embodiments, the invention relates to conjugates comprising an anti-Factor D antibody, wherein the unconjugated Fab fragment of the antibody has at least Half-lives of 3, 5, 7, 10 or 12 days. In another embodiment, the present invention is directed to a conjugate comprising a humanized anti-Factor D antibody, wherein the unconjugated Fab fragment of said antibody is administered to a mammalian eye (e.g., a human eye) via a single intravitreal injection. ) for at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 days after inhibiting alternative pathway (AP) complement activation. In another embodiment, the present invention is directed to a conjugate comprising a humanized anti-Factor D antibody, wherein the unconjugated Fab fragment of said antibody inhibits alternative pathway (AP) complement activation at a concentration obtained via a single Intravitreal injection is maintained in retinal tissue for at least 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 days after administration to a mammalian eye (eg, a human). In another embodiment, the present invention is directed to a conjugate comprising a humanized anti-Factor D antibody, wherein the unconjugated Fab fragment of said antibody inhibits alternative pathway (AP) complement activation at a concentration obtained via a single Intravitreal injections are maintained in the vitreous humor for at least 80, 85, 90, 95, 100, 105, 110, or 115 days after administration to a mammalian eye (eg, a human).
抗-因子D抗体或抗体变体-聚合物缀合物的构建Construction of Anti-Factor D Antibody or Antibody Variant-Polymer Conjugates
a.多臂聚合物a. Multi-arm polymer
在一些方面，能够通过将本文描述的抗-因子D抗体或抗体变体通过缀合抗体或其变体与多臂聚合物进行衍生化来制备本发明的缀合物。应当理解，提供具有所需尺寸的缀合物或具有如本文描述的所选平均分子量的任何多臂聚合物适用于构建本发明的抗体-聚合物缀合物。In some aspects, the conjugates of the invention can be prepared by derivatizing an anti-Factor D antibody or antibody variant described herein by conjugating the antibody or variant thereof with a multi-armed polymer. It will be appreciated that any multi-armed polymer that provides a conjugate of the desired size or with a selected average molecular weight as described herein is suitable for use in the construction of the antibody-polymer conjugates of the invention.
许多聚合物适用于药物。参见，例如，Davis等人,Biomedical Polymers:Polymeric Materials and Pharmaceuticals for Biomedical Use,pp.441-451(1980)。在本发明的所有实施方案中，将非蛋白质聚合物用于形成本发明的缀合物。非蛋白质聚合物通常是亲水性合成聚合物，即在自然界中不以其它方式发现的聚合物。然而，天然存在并且通过重组或体外方法产生的聚合物也可以是有用的，如从天然来源分离的聚合物也是有用的。Many polymers are suitable for use in pharmaceuticals. See, eg, Davis et al., Biomedical Polymers: Polymeric Materials and Pharmaceuticals for Biomedical Use, pp. 441-451 (1980). In all embodiments of the invention, non-proteinaceous polymers are used to form the conjugates of the invention. Non-proteinaceous polymers are generally hydrophilic synthetic polymers, ie polymers not otherwise found in nature. However, polymers that occur in nature and are produced by recombinant or in vitro methods may also be useful, such as polymers isolated from natural sources.
在一些方面，抗-因子D抗体或抗体变体通过将抗体或其变体缀合(例如，共价连接)至多臂多元醇来进行衍生化。因此，在一些实施方案中，本发明涉及包含与一种或多种多臂多元醇共价连接的本文公开的一种或多种抗-因子D抗体或抗体变体的缀合物。采用的多元醇可以是任何水溶性聚(氧化烯)聚合物，并且能够具有直链或支链。合适的多元醇包括在一个或多个羟基位置处被化学基团(诸如，具有一至四个碳的烷基)取代的那些多元醇。典型地，多元醇是聚(亚烷基二醇)，诸如聚乙二醇(PEG)，并且因此为了便于描述，其余的讨论涉及其中采用的多元醇是PEG的示例性实施方案，并且将多元醇缀合至多肽的过程称为“PEG化”。然而，本领域技术人员将认识到可以使用本文描述的用于PEG的缀合技术来利用其它多元醇，诸如例如聚(丙二醇)和聚乙二醇-聚丙二醇共聚物。In some aspects, an anti-Factor D antibody or antibody variant is derivatized by conjugating (eg, covalently linking) the antibody or variant thereof to a multi-armed polyol. Accordingly, in some embodiments, the present invention is directed to conjugates comprising one or more anti-Factor D antibodies or antibody variants disclosed herein covalently linked to one or more multi-armed polyols. The polyol employed can be any water-soluble poly(oxyalkylene) polymer and can have straight or branched chains. Suitable polyols include those substituted at one or more hydroxyl positions with chemical groups such as alkyl groups having one to four carbons. Typically, the polyol is a poly(alkylene glycol), such as polyethylene glycol (PEG), and thus for ease of description, the remainder of the discussion refers to exemplary embodiments in which the polyol employed is PEG, and the polyol The process of conjugating an alcohol to a polypeptide is called "PEGylation." However, those skilled in the art will recognize that other polyols can be utilized using the conjugation techniques described herein for PEG, such as, for example, poly(propylene glycol) and polyethylene glycol-polypropylene glycol copolymers.
用于形成本发明的缀合物的多元醇是多臂多元醇。如本文所使用的，“多臂多元醇”是指包含与至少两个臂连接的核心结构的多元醇。多臂多元醇可以是例如二聚体(两个臂)，四聚体(四个臂)，六聚体(六个臂)，八聚体(八个臂)等。在一些方面，多臂多元醇是多臂PEG。The polyols used to form the conjugates of the invention are multi-armed polyols. As used herein, "multi-armed polyol" refers to a polyol comprising a core structure connected to at least two arms. Multi-armed polyols can be, for example, dimers (two arms), tetramers (four arms), hexamers (six arms), octamers (eight arms), and the like. In some aspects, the multi-armed polyol is a multi-armed PEG.
用于抗-因子D抗体和抗体变体的PEG化中的多臂PEG的重均分子量可以变化，并且通常可以在约500至约300,000道尔顿(D)的范围内。在一些实施方案中，多臂PEG的重均分子量是约1,000至约100,000D，并且在一些实施方案中是约20,000至约60,000D。在一些实施方案中，使用重均分子量为约40,000D的多臂PEG进行PEG化。The weight average molecular weight of the multi-arm PEG used in the PEGylation of anti-Factor D antibodies and antibody variants can vary, and typically can range from about 500 to about 300,000 Daltons (D). In some embodiments, the weight average molecular weight of the multi-armed PEG is from about 1,000 to about 100,000 D, and in some embodiments from about 20,000 to about 60,000 D. In some embodiments, PEGylation is performed using a multi-armed PEG with a weight average molecular weight of about 40,000D.
用于PEG化蛋白质的多种方法在本领域中是已知的。产生与PEG缀合的蛋白质的具体方法包括美国专利号4,179,337、美国专利号4,935,465和美国专利号5,849,535中描述的方法，这些文献全部通过引用整体结合于此。通常，蛋白质经由蛋白质的一个或多个氨基酸残基与聚合物上的末端反应性基团共价结合。具有一个或多个反应性基团的聚合物在本文中被称为活化或官能化聚合物(例如，官能化PEG)。反应性基团选择性地与抗体或抗体变体上的游离巯基或氨基或其它反应性基团反应。多臂PEG聚合物可以以随机方式或位点特异性方式偶联至抗体或抗体变体上的巯基或氨基或其它反应性基团。然而，应当理解，为了获得最佳结果，所选择的反应性基团的类型和量以及所采用的聚合物的类型将取决于用于限制并且优选基本上防止反应性基团与抗体上的过多活性基团反应的特定抗体或抗体变体。由于在一些情况下不可能充分地限制或防止这种情形，取决于抗体浓度，可以采用通常约0.05至约1000摩尔或者在一些实施方案中约0.05至约200摩尔的官能化聚合物/摩尔抗体。每摩尔抗体的官能化聚合物的最终量是保持最佳活性的平衡，而如果可能的话，同时优化抗体的玻璃体液、视网膜和/或房水半衰期。Various methods for PEGylating proteins are known in the art. Specific methods for producing proteins conjugated to PEG include those described in US Patent No. 4,179,337, US Patent No. 4,935,465, and US Patent No. 5,849,535, all of which are hereby incorporated by reference in their entirety. Typically, the protein is covalently bound to a terminal reactive group on the polymer via one or more amino acid residues of the protein. A polymer with one or more reactive groups is referred to herein as an activated or functionalized polymer (eg, functionalized PEG). Reactive groups selectively react with free sulfhydryl or amino groups or other reactive groups on the antibody or antibody variant. Multi-armed PEG polymers can be coupled to sulfhydryl or amino groups or other reactive groups on the antibody or antibody variant in a random or site-specific manner. However, it should be understood that for best results the type and amount of reactive groups selected and the type of polymer employed will depend on the methods used to limit and preferably substantially prevent excessive interaction of the reactive groups with the antibody. Specific antibodies or antibody variants reactive with multiple reactive groups. Since in some cases it may not be possible to adequately limit or prevent this situation, depending on the concentration of the antibody, generally from about 0.05 to about 1000 moles or in some embodiments from about 0.05 to about 200 moles of functionalized polymer per mole of antibody can be employed . The final amount of functionalized polymer per mole of antibody is a balance to maintain optimal activity while, if possible, optimizing the vitreous humor, retinal and/or aqueous humor half-life of the antibody.
虽然残基可以是抗体或抗体变体上的任何反应性氨基酸，诸如N-末端氨基酸基团，但是在一些实施方案中，反应性氨基酸是半胱氨酸，其经由其游离硫醇基团与官能化聚合物的反应性基团连接，如例如在WO 99/03887、WO 94/12219、WO 94/22466、美国专利号5,206,344、美国专利号5,166,322和美国专利号5,206,344中所示的，这些文献全部通过引用整体结合于此。在这样的实施方案中，聚合物可以包含至少一个末端反应性基团，所述反应性基团能够与亲本抗体上的一个或多个游离巯基或硫醇基团特异性反应。其中，这种基团包括但不限于，马来酰亚胺、巯基、硫醇、三氟甲磺酸酯、甲苯磺酸酯、氮丙啶、环氧化物、吡啶基二硫化物、琥珀酰亚胺酯、-NH2、醛、卤代乙酸酯、卤代乙酰胺和碳酸对-硝基苯酯。可以使用适用于所选的偶联系统的化学的任何方案将聚合物偶联至亲本抗体，诸如美国专利号4,179,337、美国专利号7,122,636以及Jevsevar等人,Biotech J.,Vol.5,pp.113-128(2010)中所描述的方案和系统。备选地，反应性氨基酸可以是赖氨酸，其经由其游离ε-氨基连接至官能化聚合物的反应性基团(参见例如WO 93/00109，其通过引用结合于此)，或者谷氨酸或天冬氨酸，其经由酰胺键与聚合物连接。然后，聚合物的反应性基团可以与例如蛋白质的α(alpha)和ε(epsilon)胺或巯基反应以形成共价键。应当理解，本发明不限于利用抗体或抗体片段与聚合物之间的任何特定类型的连接的缀合物。Although the residue can be any reactive amino acid on an antibody or antibody variant, such as the N-terminal amino acid group, in some embodiments, the reactive amino acid is cysteine, which interacts with Reactive group attachment of functionalized polymers, as shown for example in WO 99/03887, WO 94/12219, WO 94/22466, U.S. Patent No. 5,206,344, U.S. Patent No. 5,166,322 and U.S. Patent No. 5,206,344, which All are hereby incorporated by reference in their entirety. In such embodiments, the polymer may comprise at least one terminal reactive group capable of specifically reacting with one or more free sulfhydryl or thiol groups on the parent antibody. Among them, such groups include, but are not limited to, maleimide, mercapto, thiol, triflate, tosylate, aziridine, epoxide, pyridyl disulfide, succinyl Imidoesters,-NH2 , aldehydes, haloacetates, haloacetamides, and p-nitrophenyl carbonate. The polymer can be coupled to the parent antibody using any protocol suitable for the chemistry of the chosen coupling system, such as U.S. Patent No. 4,179,337, U.S. Patent No. 7,122,636, and Jevsevar et al., Biotech J., Vol.5, pp. - Protocol and system described in 128 (2010). Alternatively, the reactive amino acid may be lysine, which is linked via its free ε-amino group to the reactive group of the functionalized polymer (see e.g. WO 93/00109, which is hereby incorporated by reference), or glutamine acid or aspartic acid, which is linked to the polymer via an amide bond. The reactive groups of the polymer can then react with, for example, the alpha (alpha) and epsilon (epsilon) amine or sulfhydryl groups of the protein to form a covalent bond. It should be understood that the invention is not limited to conjugates utilizing any particular type of linkage between the antibody or antibody fragment and the polymer.
用于制备本发明缀合物的合适的官能化多臂PEG可以通过许多常规反应产生。例如，PEG的N-羟基琥珀酰亚胺酯(M-NHS-PEG)可以根据Buckmann和Merr,Makromol.Chem.,Vol.182,pp.1379-1384(1981)的方法，通过与N,N’-二环己基碳二亚胺(DCC)和N-羟基琥珀酰亚胺(NHS)反应由PEG-单甲基醚制备。另外，例如，通过与亚硫酰溴反应形成PEG-Br，然后用过量氨进行氨解以形成PEG-NH2，可以将PEG末端羟基转化为氨基。然后，可以使用标准偶联剂(诸如伍德沃德氏试剂K(Woodward’s Reagent K))将PEG-NH2缀合至感兴趣的抗体或抗体变体。此外，可以例如通过用MnO2进行氧化将PEG末端-CH2OH基团转化为醛基。醛基可以通过用试剂(诸如氰基硼氢化物)进行还原性烷基化而与抗体或抗体变体缀合。Suitable functionalized multi-armed PEGs for use in preparing conjugates of the invention can be produced by a number of routine reactions. For example, the N-hydroxysuccinimide ester of PEG (M-NHS-PEG) can be prepared according to Buckmann and Merr, Makromol.Chem., Vol.182, pp.1379-1384 (1981), by combining The reaction of '-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) was prepared from PEG-monomethyl ether. Alternatively, PEG terminal hydroxyl groups can be converted to amino groups, for example, by reaction with thionyl bromide to form PEG-Br, followed by ammonolysis with excess ammonia to form PEG-NH2 . PEG-NH2 can then be conjugated to the antibody or antibody variant of interest using standard coupling reagents such as Woodward's Reagent K. Furthermore, PEG terminal-CH2OH groups can be converted to aldehyde groups, for example by oxidation withMnO2 . Aldehyde groups can be conjugated to antibodies or antibody variants by reductive alkylation with reagents such as cyanoborohydride.
在一些实施方案中，用于制备本发明缀合物的多臂PEG具有通式(I)的结构：In some embodiments, the multi-armed PEG used to prepare the conjugates of the invention has the structure of general formula (I):
其中各m表示多元醇(PEG)的特定臂的长度或尺寸，并且独立地是约45至约1000、约3至约250、或约50至约200、或约100至约150的整数；并且n是约1至约10的整数。wherein each m represents the length or size of a particular arm of a polyol (PEG), and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10.
在一些实施方案中，多臂PEG具有通式(I)的结构，其中n是1，并且多臂PEG是四聚体。在另一个实施方案中，多臂PEG具有通式(I)的结构，其中n是2，并且多臂PEG是六聚体。在另一个实施方案中，多臂PEG具有通式(I)的结构，其中n是3，并且多臂PEG是八聚体。In some embodiments, the multi-armed PEG has the structure of general formula (I), wherein n is 1, and the multi-armed PEG is a tetramer. In another embodiment, the multi-armed PEG has the structure of general formula (I), wherein n is 2, and the multi-armed PEG is a hexamer. In another embodiment, the multi-armed PEG has the structure of general formula (I), wherein n is 3, and the multi-armed PEG is an octamer.
在另一个方面，用于制备本发明缀合物的多臂PEG具有通式(II)的结构：In another aspect, the multi-armed PEG used to prepare the conjugates of the invention has the general formula (II):
其中各m表示多元醇(PEG)的特定臂的长度或尺寸，并且独立地是约45至约1000、约3至约250、或约50至约200、或约100至约150的整数；并且n是约1至约10的整数。wherein each m represents the length or size of a particular arm of a polyol (PEG), and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10.
在一些实施方案中，多臂PEG具有通式(II)的结构，其中n是2，并且多臂PEG是四聚体。在另一个实施方案中，多臂PEG具有通式(II)的结构，其中n是4，并且多臂PEG是六聚体。在另一个实施方案中，多臂PEG具有通式(II)的结构，其中n是6，并且多臂PEG是八聚体。In some embodiments, the multi-armed PEG has the structure of general formula (II), wherein n is 2, and the multi-armed PEG is a tetramer. In another embodiment, the multi-armed PEG has the structure of general formula (II), wherein n is 4, and the multi-armed PEG is a hexamer. In another embodiment, the multi-armed PEG has the structure of general formula (II), wherein n is 6, and the multi-armed PEG is an octamer.
在另一个方面，用于制备本发明缀合物的多臂PEG具有通式(III)的结构：In another aspect, the multi-armed PEG used to prepare the conjugates of the invention has the structure of general formula (III):
其中各m表示多元醇(PEG)的特定臂的长度或尺寸，并且独立地是约45至约1000、约3至约250、或约50至约200、或约100至约150的整数；并且n是约1至约10的整数。wherein each m represents the length or size of a particular arm of a polyol (PEG), and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10.
在一些实施方案中，多臂PEG具有通式(III)的结构，其中n是2，并且多臂PEG是四聚体。在另一个实施方案中，多臂PEG具有通式(III)的结构，其中n是4，并且多臂PEG是六聚体。在另一个实施方案中，多臂PEG具有通式(III)的结构，其中n是6，并且多臂PEG是八聚体。In some embodiments, the multi-armed PEG has the structure of general formula (III), wherein n is 2, and the multi-armed PEG is a tetramer. In another embodiment, the multi-armed PEG has the structure of general formula (III), wherein n is 4, and the multi-armed PEG is a hexamer. In another embodiment, the multi-armed PEG has the structure of general formula (III), wherein n is 6, and the multi-armed PEG is an octamer.
在另一个方面，用于制备本发明缀合物的多臂PEG具有通式(IV)的结构：In another aspect, the multi-armed PEG used to prepare the conjugates of the invention has the structure of general formula (IV):
其中各m表示多元醇(PEG)的特定臂的长度或尺寸，并且独立地是约45至约1000、约3至约250、或约50至约200、或约100至约150的整数。wherein each m represents the length or size of a particular arm of the polyol (PEG) and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150.
具有通式(I)至(IV)中任一个的结构的多臂PEG可以被官能化以例如使用如上描述的任一个技术连接适于与抗体(例如抗体片段)反应或缀合的末端反应性基团，以产生官能化多臂PEG。然而，在其它实施方案中，多臂PEG可以经由多官能交联剂共价连接至抗-因子D抗体或抗体变体，所述多官能交联剂与PEG以及待连接的抗体或抗体片段的一个或多个氨基酸残基反应，如例如，美国专利号7,122,636中所描述的，该文献通过引用整体结合于此。A multi-armed PEG having a structure of any of the general formulas (I) to (IV) can be functionalized, for example, using any of the techniques described above to attach end-reactive compounds suitable for reaction or conjugation with antibodies (e.g., antibody fragments). groups to generate functionalized multi-armed PEGs. However, in other embodiments, the multi-armed PEG can be covalently linked to the anti-Factor D antibody or antibody variant via a multifunctional cross-linking agent that is compatible with the PEG and the antibody or antibody fragment to be linked. One or more amino acid residues are reacted as described, eg, in US Pat. No. 7,122,636, which is hereby incorporated by reference in its entirety.
在其它方面，用于制备本发明缀合物的多臂PEG是包含至少一个末端反应性基团的官能化多臂PEG。末端反应性基团可以直接缀合至抗-因子D抗体或抗体变体以形成本发明的缀合物。在一些实施方案中，官能化多臂PEG具有通式(Ia)的结构：In other aspects, the multi-arm PEG used to prepare the conjugates of the invention is a functionalized multi-arm PEG comprising at least one terminal reactive group. The terminal reactive group can be directly conjugated to an anti-Factor D antibody or antibody variant to form a conjugate of the invention. In some embodiments, the functionalized multi-armed PEG has the general formula (Ia):
其中各m表示多元醇(PEG)的特定臂的长度或尺寸，并且独立地是约45至约1000、约3至约250、或约50至约200、或约100至约150的整数；并且n是约1至约10的整数；各R1独立地是不存在或是连接基团；并且各R2独立地是氢或末端反应性基团；其中至少一个R2是末端反应性基团。在一些实施方案中，R2独立地选自由以下组成的组：硫醇反应性基团、氨基反应性基团及其组合。wherein each m represents the length or size of a particular arm of a polyol (PEG), and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10; eachR is independently absent or a linking group; and eachR is independently hydrogen or a terminal reactive group; wherein at leastone R is a terminal reactive group . In some embodiments,R2 is independently selected from the group consisting of thiol reactive groups, amino reactive groups, and combinations thereof.
在一些实施方案中，官能化多臂PEG具有通式(Ia)的结构，其中n是1至3的整数。在一些实施方案中，官能化多臂PEG具有通式(Ia)的结构，其中n是1，并且多臂PEG是四聚体。在另一个实施方案中，官能化多臂PEG具有通式(Ia)的结构，其中n是2，并且多臂PEG是六聚体。在另一个实施方案中，官能化多臂PEG具有通式(Ia)的结构，其中n是3，并且多臂PEG是八聚体。在这样的实施方案中，八聚体具有通式(Ib)的结构：In some embodiments, the functionalized multi-armed PEG has the structure of formula (Ia), wherein n is an integer from 1 to 3. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (Ia), wherein n is 1, and the multi-arm PEG is a tetramer. In another embodiment, the functionalized multi-arm PEG has the structure of general formula (Ia), wherein n is 2, and the multi-arm PEG is a hexamer. In another embodiment, the functionalized multi-arm PEG has the structure of general formula (Ia), wherein n is 3 and the multi-arm PEG is an octamer. In such an embodiment, the octamer has the structure of general formula (Ib):
其中m、R1、R2是如本文中所定义的。特别地，在一个实施方案中，各m表示多元醇(PEG)的特定臂的长度或尺寸，并且独立地是约45至约1000、约3至约250、或约50至约200、或约100至约150的整数；各R1独立地是不存在或是连接基团；并且各R2独立地是氢或末端反应性基团；其中至少一个R2是末端反应性基团。在一些实施方案中，R2独立地选自由以下组成的组：硫醇反应性基团、氨基反应性基团及其组合。wherein m, R1 , R2 are as defined herein. In particular, in one embodiment, each m represents the length or size of a particular arm of a polyol (PEG), and is independently about 45 to about 1000, about 3 to about 250, or about 50 to about 200, or about an integer from 100 to about 150; eachR is independently absent or a linking group; and eachR is independently hydrogen or a terminal reactive group; wherein at least oneR is a terminal reactive group. In some embodiments,R2 is independently selected from the group consisting of thiol reactive groups, amino reactive groups, and combinations thereof.
具有通式(Ib)的结构的多臂PEG具有三季戊四醇(TP)核心结构，并且在本文中也称为TP八聚体。A multi-armed PEG having a structure of general formula (Ib) has a tripentaerythritol (TP) core structure and is also referred to herein as a TP octamer.
在一些实施方案中，官能化多臂PEG具有通式(Ia)或(Ib)的结构，其中各R1当存在时是相同或不同的，并且R1和R2当连接一起时选自由以下组成的组：In some embodiments, the functionalized multi-armed PEG has a structure of general formula (Ia) or (Ib), wherein each R1 when present is the same or different, and R1 and R2 when linked together are selected from the group consisting of Composed of groups:
及其组合；其中各i独立地是0-10的整数；j是0-10的整数；并且R2是如本文定义的。在一些实施方案中，各R1是连接基团。and combinations thereof; wherein each i is independently an integer from 0-10; j is an integer from 0-10; andR is as defined herein. In some embodiments, each R1 is a linking group.
在一些实施方案中，官能化多臂PEG具有通式(Ia)或(Ib)的结构，其中R1和R2当连接一起时是其中i、j和R2是如本文定义的。在一些实施方案中，R1和R2当连接一起时是其中i是2；j是2或3，并且R2是如本文定义的。In some embodiments, the functionalized multi-armed PEG has the structure of formula (Ia) or (Ib), whereinR andR when linked together are wherein i, j andR are as defined herein. In some embodiments, R1 and R2 when linked together are wherein i is 2; j is 2 or 3, andR is as defined herein.
在一些方面，官能化多臂PEG具有通式(Ia)或(Ib)的结构，各R2独立地选自由以下组成的组：马来酰亚胺、巯基、硫醇、三氟甲磺酸酯、甲苯磺酸酯、氮丙啶、环氧化物、吡啶基二硫化物、琥珀酰亚胺酯、-NH2、醛、卤代乙酸酯、卤代乙酰胺和碳酸对-硝基苯酯。在一些实施方案中，各R2独立地是选自由以下组成的组的卤代乙酸酯：溴代乙酸酯、碘代乙酸酯、氯代乙酸酯及其组合。在一些实施方案中，各R2独立地是选自由以下组成的组的卤代乙酰胺：溴代乙酰胺、碘代乙酰胺、氯代乙酰胺及其组合。在一些实施方案中，R2是马来酰亚胺。In some aspects, the functionalized multi-armed PEG has the structure of formula (Ia) or (Ib), eachR2 is independently selected from the group consisting of: maleimide, thiol, thiol, trifluoromethanesulfonic acid Esters, Tosylates, Aziridines, Epoxides, Pyridyl Disulfides, Succinimidyl Esters, -NH2 , Aldehydes, Haloacetates, Haloacetamides, and p-Nitrobenzene Carbonate ester. In some embodiments, eachR2 is independently a haloacetate selected from the group consisting of bromoacetate, iodoacetate, chloroacetate, and combinations thereof. In some embodiments, eachR2 is independently a haloacetamide selected from the group consisting of bromoacetamide, iodoacetamide, chloroacetamide, and combinations thereof. In some embodiments, R2 is maleimide.
在一些实施方案中，官能化多臂PEG具有通式(Ia)或(Ib)的结构，其中各R2是马来酰亚胺。在一些实施方案中，官能化多臂PEG具有通式(Ia)或(Ib)的结构，其中R1和R2当连接一起时是其中i和j是如本文定义的。在一个实施方案中，官能化多臂PEG具有通式(Ia)或(Ib)的结构，其中R1和R2当连接一起时是其中i是2并且j是2。In some embodiments, the functionalized multi-armed PEG has the structure of formula (Ia) or (Ib), wherein each R2 is maleimide. In some embodiments, the functionalized multi-armed PEG has the structure of formula (Ia) or (Ib), whereinR andR when linked together are wherein i and j are as defined herein. In one embodiment, the functionalized multi-armed PEG has the structure of general formula (Ia) or (Ib), whereinR andR when linked together are where i is 2 and j is 2.
在另一个方面，用于制备本发明缀合物的官能化多臂PEG具有通式(IIa)的结构：In another aspect, the functionalized multi-armed PEG used to prepare the conjugates of the invention has the general formula (IIa):
其中各m表示多元醇(PEG)的特定臂的长度或尺寸，并且独立地是约45至约1000、约3至约250、或约50至约200、或约100至约150的整数；并且n是约1至约10的整数；各R1独立地是不存在或是连接基团；并且各R2独立地是氢或末端反应性基团；其中至少一个R2是末端反应性基团。在一些实施方案中，R2独立地选自由以下组成的组：硫醇反应性基团、氨基反应性基团及其组合。wherein each m represents the length or size of a particular arm of a polyol (PEG), and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10; eachR is independently absent or a linking group; and eachR is independently hydrogen or a terminal reactive group; wherein at leastone R is a terminal reactive group . In some embodiments,R2 is independently selected from the group consisting of thiol reactive groups, amino reactive groups, and combinations thereof.
在一些实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中n是2至6的整数。在一些实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中n是3。在一个实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中n是2，并且多臂PEG是四聚体。在另一个实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中n是4，并且多臂PEG是六聚体。在另一个实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中n是6，并且多臂PEG是八聚体。具有通式(IIa)的结构的八聚体具有六甘油(HG)核心结构，并且在本文中也称为HG八聚体。In some embodiments, the functionalized multi-armed PEG has the structure of formula (IIa), wherein n is an integer from 2 to 6. In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IIa), wherein n is 3. In one embodiment, the functionalized multi-armed PEG has the structure of general formula (IIa), wherein n is 2, and the multi-armed PEG is a tetramer. In another embodiment, the functionalized multi-arm PEG has the structure of general formula (IIa), wherein n is 4 and the multi-arm PEG is a hexamer. In another embodiment, the functionalized multi-arm PEG has the structure of general formula (IIa), wherein n is 6, and the multi-arm PEG is an octamer. Octamers having a structure of general formula (IIa) have a hexaglycerol (HG) core structure and are also referred to herein as HG octamers.
在一些实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中各R1当存在时是相同或不同的，并且R1和R2当连接一起时选自由以下组成的组：In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IIa), wherein each R1 when present is the same or different, and R1 and R2 when linked together are selected from the group consisting of:
及其组合；其中各i独立地是0-10的整数；j是0-10的整数；并且R2是如本文定义的。在一些实施方案中，各R1是连接基团。and combinations thereof; wherein each i is independently an integer from 0-10; j is an integer from 0-10; andR is as defined herein. In some embodiments, each R1 is a linking group.
在一些实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中R1和R2当连接一起时是其中i、j和R2是如本文定义的。在一些实施方案中，R1和R2当连接一起时是其中i是2；j是2或3，并且R2是如本文定义的。In some embodiments, the functionalized multi-armed PEG has the structure of formula (IIa), wherein RandR when linked together are wherein i, j andR are as defined herein. In some embodiments, R1 and R2 when linked together are wherein i is 2; j is 2 or 3, andR is as defined herein.
在一些方面，官能化多臂PEG具有通式(IIa)的结构，各R2独立地选自由以下组成的组：马来酰亚胺、巯基、硫醇、三氟甲磺酸酯、甲苯磺酸酯、氮丙啶、环氧化物、吡啶基二硫化物、琥珀酰亚胺酯、-NH2、醛、卤代乙酸酯、卤代乙酰胺和碳酸对-硝基苯酯。在一些实施方案中，各R2独立地是选自由以下组成的组的卤代乙酸酯：溴代乙酸酯、碘代乙酸酯、氯代乙酸酯及其组合。在一些实施方案中，各R2独立地是选自由以下组成的组的卤代乙酰胺：溴代乙酰胺、碘代乙酰胺、氯代乙酰胺及其组合。在一些实施方案中，R2是马来酰亚胺。In some aspects, the functionalized multi-armed PEG has the structure of general formula (IIa),each R is independently selected from the group consisting of: maleimide, thiol, thiol, triflate, tosylate esters, aziridines, epoxides, pyridyl disulfides, succinimidyl esters,-NH2 , aldehydes, haloacetates, haloacetamides, and p-nitrophenyl carbonate. In some embodiments, eachR2 is independently a haloacetate selected from the group consisting of bromoacetate, iodoacetate, chloroacetate, and combinations thereof. In some embodiments, eachR2 is independently a haloacetamide selected from the group consisting of bromoacetamide, iodoacetamide, chloroacetamide, and combinations thereof. In some embodiments, R2 is maleimide.
在一些实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中各R2是马来酰亚胺。在一些实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中R1和R2当连接一起时是其中i和j是如本文定义的。在一些实施方案中，官能化多臂PEG具有通式(IIa)的结构，其中R1和R2当连接一起时是其中i是2并且j是2。In some embodiments, the functionalized multi-armed PEG has the structure of Formula (IIa), wherein each R2 is maleimide. In some embodiments, the functionalized multi-armed PEG has the structure of formula (IIa), wherein RandR when linked together are wherein i and j are as defined herein. In some embodiments, the functionalized multi-armed PEG has the structure of formula (IIa), wherein RandR when linked together are where i is 2 and j is 2.
在另一个实施方案中，官能化多臂PEG具有通式(IIIa)的结构：In another embodiment, the functionalized multi-armed PEG has the general formula (Ilia):
其中各m表示多元醇(PEG)的特定臂的长度或尺寸，并且独立地是约45至约1000、约3至约250、或约50至约200、或约100至约150的整数；并且n是约1至约10的整数；各R1独立地是不存在或是连接基团；并且各R2独立地是氢或末端反应性基团；其中至少一个R2是末端反应性基团。在一些实施方案中，R2独立地选自由以下组成的组：硫醇反应性基团、氨基反应性基团及其组合。wherein each m represents the length or size of a particular arm of a polyol (PEG), and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; and n is an integer from about 1 to about 10; eachR is independently absent or a linking group; and eachR is independently hydrogen or a terminal reactive group; wherein at leastone R is a terminal reactive group . In some embodiments,R2 is independently selected from the group consisting of thiol reactive groups, amino reactive groups, and combinations thereof.
在一些实施方案中，官能化多臂PEG具有通式(IIIa)的结构，其中n是2至6的整数。在一些实施方案中，官能化多臂PEG具有通式(IIIa)的结构，其中n是2，并且多臂PEG是四聚体。在另一个实施方案中，官能化多臂PEG具有通式(IIIa)的结构，其中n是4，并且多臂PEG是六聚体。在另一个实施方案中，官能化多臂PEG具有通式(IIIa)的结构，其中n是6，并且多臂PEG是八聚体。具有通式(IIIa)的结构的八聚体具有六甘油(HGEO)核心结构，并且在本文中也称为HGEO八聚体。In some embodiments, the functionalized multi-armed PEG has the structure of formula (Ilia), wherein n is an integer from 2 to 6. In some embodiments, the functionalized multi-arm PEG has the structure of general formula (Ilia), wherein n is 2, and the multi-arm PEG is a tetramer. In another embodiment, the functionalized multi-arm PEG has the structure of general formula (Ilia), wherein n is 4 and the multi-arm PEG is a hexamer. In another embodiment, the functionalized multi-arm PEG has the structure of general formula (Ilia), wherein n is 6, and the multi-arm PEG is an octamer. Octamers having the structure of general formula (Ilia) have a hexaglycerol (HGEO) core structure and are also referred to herein as HGEO octamers.
在一些实施方案中，官能化多臂PEG具有通式(IIIa)的结构，其中各R1当存在时是相同或不同的，并且R1和R2当连接一起时选自由以下组成的组：In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IIIa), wherein each R1 when present is the same or different, and R1 and R2 when linked together are selected from the group consisting of:
及其组合；其中各i独立地是0-10的整数；j是0-10的整数；并且R2是如本文定义的。在一些实施方案中，各R1是连接基团。and combinations thereof; wherein each i is independently an integer from 0-10; j is an integer from 0-10; andR is as defined herein. In some embodiments, each R1 is a linking group.
在一些实施方案中，官能化多臂PEG具有通式(IIIa)的结构，其中R1和R2当连接一起时是其中i、j和R2是如本文定义的。在一些实施方案中，R1和R2当连接一起时是其中i是2；j是2或3，并且R2是如本文定义的。In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IIIa), wherein RandR when linked together are wherein i, j andR are as defined herein. In some embodiments, R1 and R2 when linked together are wherein i is 2; j is 2 or 3, andR is as defined herein.
在一些方面，官能化多臂PEG具有通式(IIIa)的结构，各R2独立地选自由以下组成的组：马来酰亚胺、巯基、硫醇、三氟甲磺酸酯、甲苯磺酸酯、氮丙啶、环氧化物、吡啶基二硫化物、琥珀酰亚胺酯、-NH2、醛、卤代乙酸酯、卤代乙酰胺和碳酸对-硝基苯酯。在一些实施方案中，各R2独立地是选自由以下组成的组的卤代乙酸酯：溴代乙酸酯、碘代乙酸酯、氯代乙酸酯及其组合。在一些实施方案中，各R2独立地是选自由以下组成的组的卤代乙酰胺：溴代乙酰胺、碘代乙酰胺、氯代乙酰胺及其组合。在一些实施方案中，R2是马来酰亚胺。In some aspects, the functionalized multi-armed PEG has the structure of general formula (IIIa),each R is independently selected from the group consisting of: maleimide, thiol, thiol, triflate, tosylate esters, aziridines, epoxides, pyridyl disulfides, succinimidyl esters,-NH2 , aldehydes, haloacetates, haloacetamides, and p-nitrophenyl carbonate. In some embodiments, eachR2 is independently a haloacetate selected from the group consisting of bromoacetate, iodoacetate, chloroacetate, and combinations thereof. In some embodiments, eachR2 is independently a haloacetamide selected from the group consisting of bromoacetamide, iodoacetamide, chloroacetamide, and combinations thereof. In some embodiments, R2 is maleimide.
在一些实施方案中，官能化多臂PEG具有通式(IIIa)的结构，其中各R2是马来酰亚胺。在一些实施方案中，官能化多臂PEG具有通式(IIIa)的结构，其中R1和R2当连接一起时是其中i和j是如本文定义的。在一些实施方案中，官能化多臂PEG具有通式(IIIa)的结构，其中R1和R2当连接一起时是其中i是3并且j是2。In some embodiments, the functionalized multi-armed PEG has the structure of formula (IIIa), wherein each R2 is maleimide. In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IIIa), wherein RandR when linked together are wherein i and j are as defined herein. In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IIIa), wherein RandR when linked together are where i is 3 and j is 2.
在另一个实施方案中，官能化多臂PEG具有通式(IVa)的结构：In another embodiment, the functionalized multi-armed PEG has the general formula (IVa):
其中各m表示多元醇(PEG)的特定臂的长度或尺寸，并且独立地是约45至约1000、约3至约250、或约50至约200、或约100至约150的整数；各R1独立地是不存在或是连接基团；并且各R2独立地是氢或末端反应性基团；其中至少一个R2是末端反应性基团。在一些实施方案中，R2独立地选自由以下组成的组：硫醇反应性基团、氨基反应性基团及其组合。wherein each m represents the length or size of a particular arm of a polyol (PEG), and is independently an integer from about 45 to about 1000, from about 3 to about 250, or from about 50 to about 200, or from about 100 to about 150; each R1 is independently absent or a linking group; and each R2 is independently hydrogen or a terminal reactive group; wherein at least one R2 is a terminal reactive group. In some embodiments,R2 is independently selected from the group consisting of thiol reactive groups, amino reactive groups, and combinations thereof.
具有通式(IVa)的结构的多臂PEG具有丁二醇核心结构，并且在本文中也称为DX八聚体。A multi-armed PEG having a structure of general formula (IVa) has a butanediol core structure and is also referred to herein as a DX octamer.
在一些实施方案中，官能化多臂PEG具有通式(IVa)的结构，其中各R1当存在时是相同或不同的，并且R1和R2当连接一起时选自由以下组成的组：In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IVa), wherein each R1 when present is the same or different, and R1 and R2 when linked together are selected from the group consisting of:
及其组合；其中各i独立地是0-10的整数；j是0-10的整数；并且R2是如本文定义的。在一些实施方案中，各R1是连接基团。and combinations thereof; wherein each i is independently an integer from 0-10; j is an integer from 0-10; andR is as defined herein. In some embodiments, each R1 is a linking group.
在一些实施方案中，官能化多臂PEG具有通式(IVa)的结构，其中R1和R2当连接一起时是其中i、j和R2是如本文定义的。在一些实施方案中，R1和R2当连接一起时是其中i是2；j是2或3，并且R2是如本文定义的。In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IVa), wherein RandR when linked together are wherein i, j andR are as defined herein. In some embodiments, R1 and R2 when linked together are wherein i is 2; j is 2 or 3, andR is as defined herein.
在一些方面，各R2独立地选自由以下组成的组：马来酰亚胺、巯基、硫醇、三氟甲磺酸酯、甲苯磺酸酯、氮丙啶、环氧化物、吡啶基二硫化物、琥珀酰亚胺酯、-NH2、醛、卤代乙酸酯、卤代乙酰胺和碳酸对-硝基苯酯。在一些实施方案中，各R2独立地是选自由以下组成的组的卤代乙酸酯：溴代乙酸酯、碘代乙酸酯、氯代乙酸酯及其组合。在一些实施方案中，各R2独立地是选自由以下组成的组的卤代乙酰胺：溴代乙酰胺、碘代乙酰胺、氯代乙酰胺及其组合。在一些实施方案中，R2是马来酰亚胺。In some aspects, eachR is independently selected from the group consisting of maleimide, mercapto, thiol, triflate, tosylate, aziridine, epoxide, pyridyldi Sulfides, succinimidyl esters,-NH2 , aldehydes, haloacetates, haloacetamides, and p-nitrophenyl carbonate. In some embodiments, eachR2 is independently a haloacetate selected from the group consisting of bromoacetate, iodoacetate, chloroacetate, and combinations thereof. In some embodiments, eachR2 is independently a haloacetamide selected from the group consisting of bromoacetamide, iodoacetamide, chloroacetamide, and combinations thereof. In some embodiments, R2 is maleimide.
在一些实施方案中，官能化多臂PEG具有通式(IVa)的结构，其中各R2是马来酰亚胺。在一些实施方案中，官能化多臂PEG具有通式(IVa)的结构，其中R1和R2当连接一起时是其中i和j是如本文定义的。在一些实施方案中，官能化多臂PEG具有通式(IVa)的结构，其中R1和R2当连接一起时是其中i是3并且j是2。In some embodiments, the functionalized multi-armed PEG has the structure of formula (IVa), wherein each R2 is maleimide. In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IVa), wherein RandR when linked together are wherein i and j are as defined herein. In some embodiments, the functionalized multi-armed PEG has the structure of general formula (IVa), wherein RandR when linked together are where i is 3 and j is 2.
用于制备本发明的缀合物的多臂PEG将优选具有低的PEG链(臂)长度多分散性。特别地，用于制备缀合物的多臂PEG的高多分散性可以使最终缀合物的分析变得复杂，特别是使得准确确定抗体(例如Fab)数量/PEG变得更加困难和不确定。因此，用于形成缀合物的PEG将通常具有在约1至约1.35的范围内的多分散性(使用本领域已知的方法确定)，并且在各种实施方案中将具有约1至约1.25、约1至约1.2、约1至约1.15、约1至约1.1、约1.05、或甚至约1的多分散性。The multi-armed PEGs used to prepare the conjugates of the invention will preferably have a low polydispersity of PEG chain (arm) lengths. In particular, the high polydispersity of the multi-armed PEGs used to prepare the conjugates can complicate the analysis of the final conjugates, especially making accurate determination of antibody (e.g. Fab) quantities/PEG more difficult and uncertain . Accordingly, the PEG used to form the conjugate will generally have a polydispersity (determined using methods known in the art) in the range of about 1 to about 1.35, and in various embodiments will have a polydispersity of about 1 to about 1.35. A polydispersity of 1.25, about 1 to about 1.2, about 1 to about 1.15, about 1 to about 1.1, about 1.05, or even about 1.
适用于本发明的其它官能化多臂PEG在美国专利申请公开号2011/0286956和美国专利申请公开号2015/0073155中描述，这两篇文献通过引用整体结合于此。Other functionalized multi-armed PEGs suitable for use in the present invention are described in US Patent Application Publication No. 2011/0286956 and US Patent Application Publication No. 2015/0073155, both of which are hereby incorporated by reference in their entirety.
适用于本发明的官能化多臂PEG也可以购自多个供应商。例如，JenKemTechnology,USA销售马来酰亚胺官能化的PEG八聚体(例如，8ARM(TP)-PEG-MAL和8ARM(HG)-PEG-MAL)和四聚体。NOF America Corp.也销售马来酰亚胺官能化的PEG八聚体(例如，HGEO-400MA；DX-400MA)和四聚体(例如，PTE-400MA)。这种八聚体或四聚体可以以各种分子量获得，包括40,000D的平均分子量。Functionalized multi-armed PEGs suitable for use in the present invention are also commercially available from a variety of suppliers. For example, JenKem Technology, USA sells maleimide-functionalized PEG octamers (eg, 8ARM(TP)-PEG-MAL and 8ARM(HG)-PEG-MAL) and tetramers. NOF America Corp. also sells maleimide-functionalized PEG octamers (e.g., HGEO-400MA; DX-400MA) and tetramers (eg, PTE-400MA). Such octamers or tetramers are available in various molecular weights, including an average molecular weight of 40,000D.
b.缀合物b. Conjugates
在一些实施方案中，本发明涉及包含本文公开的一种或多种抗-因子D抗体或抗体变体和一种或多种多臂多元醇的缀合物，其中所述缀合物通过将至少一个抗-因子D抗体或抗体变体共价连接至多元醇来制备。在一些实施方案中，多臂多元醇是PEG。在一些实施方案中，PEG是八聚体。在一些实施方案中，PEG具有通式(Ia)、(Ib)、(IIa)、(IIIa)或(IVa)的结构：In some embodiments, the invention relates to conjugates comprising one or more anti-Factor D antibodies or antibody variants disclosed herein and one or more multi-armed polyols, wherein the conjugates are obtained by incorporating At least one anti-Factor D antibody or antibody variant is prepared covalently linked to a polyol. In some embodiments, the multi-armed polyol is PEG. In some embodiments, PEG is an octamer. In some embodiments, PEG has the general structure of Formula (Ia), (Ib), (IIa), (IIIa), or (IVa):
本发明的缀合物的特征可以在于与各个多臂PEG缀合的抗-因子D抗体或抗体变体的数量。这在本文中称为“fab化”或“fab化的程度”。与各个PEG缀合的抗-因子D抗体或抗体变体的数量可以取决于各种因素而变化，包括：1)PEG中臂的数量；2)PEG上的末端反应性基团的数量和/或反应性；3)PEG的核心结构；和/或4)PEG化反应条件。Conjugates of the invention can be characterized by the number of anti-Factor D antibodies or antibody variants conjugated to each multi-armed PEG. This is referred to herein as "fabization" or "degree of fabization". The number of anti-Factor D antibodies or antibody variants conjugated to each PEG can vary depending on various factors including: 1) the number of arms in the PEG; 2) the number of terminal reactive groups on the PEG and/or or reactivity; 3) the core structure of PEG; and/or 4) PEGylation reaction conditions.
在一个优选的实施方案中，本发明的缀合物包含八臂PEG，其中至少一个抗-因子D抗体或抗体变体与PEG共价连接。在另一个实施方案中，本发明的缀合物包含八臂PEG，其中至少两个所述抗-因子D抗体或所述抗体变体与PEG共价连接。在另一个实施方案中，本发明的缀合物包含八臂PEG，其中至少三个抗-因子D抗体或抗体变体与PEG共价连接。在另一个实施方案中，本发明的缀合物包含八臂PEG，其中至少四个抗-因子D抗体或抗体变体与PEG共价连接。在另一个实施方案中，本发明的缀合物包含八臂PEG，其中至少五个抗-因子D抗体或抗体变体与PEG共价连接。在另一个实施方案中，本发明的缀合物包含八臂PEG，其中至少六个抗-因子D抗体或抗体变体与PEG共价连接。在另一个实施方案中，缀合物包含八臂PEG，其中至少七个抗-因子D抗体或抗体变体与PEG共价连接。在另一个实施方案中，本发明的缀合物包含八臂PEG，其中至少八个抗-因子D抗体或抗体变体与PEG共价连接。在一些实施方案中，本发明的缀合物包含八臂PEG，其中5-8个抗-因子D抗体或抗体变体与PEG共价连接。在另一个实施方案中，本发明的缀合物包含八臂PEG，其中6-8个抗-因子D抗体或抗体变体与PEG共价连接。在另一个实施方案中，本发明的缀合物包含八臂PEG，其中7-8个抗-因子D抗体或抗体变体与PEG共价连接。In a preferred embodiment, the conjugate of the invention comprises an eight-armed PEG, wherein at least one anti-Factor D antibody or antibody variant is covalently linked to the PEG. In another embodiment, the conjugate of the invention comprises an eight-armed PEG, wherein at least two of said anti-Factor D antibodies or said antibody variants are covalently linked to PEG. In another embodiment, the conjugate of the invention comprises an eight-armed PEG, wherein at least three anti-Factor D antibodies or antibody variants are covalently linked to the PEG. In another embodiment, the conjugate of the invention comprises an eight-armed PEG, wherein at least four anti-Factor D antibodies or antibody variants are covalently linked to the PEG. In another embodiment, the conjugate of the invention comprises an eight-armed PEG, wherein at least five anti-Factor D antibodies or antibody variants are covalently linked to the PEG. In another embodiment, a conjugate of the invention comprises an eight-armed PEG, wherein at least six anti-Factor D antibodies or antibody variants are covalently linked to the PEG. In another embodiment, the conjugate comprises an eight-armed PEG, wherein at least seven anti-Factor D antibodies or antibody variants are covalently linked to the PEG. In another embodiment, the conjugate of the invention comprises an eight-armed PEG, wherein at least eight anti-Factor D antibodies or antibody variants are covalently linked to the PEG. In some embodiments, the conjugates of the invention comprise an eight-armed PEG, wherein 5-8 anti-Factor D antibodies or antibody variants are covalently linked to the PEG. In another embodiment, the conjugate of the invention comprises an eight-armed PEG, wherein 6-8 anti-Factor D antibodies or antibody variants are covalently linked to the PEG. In another embodiment, the conjugate of the invention comprises an eight-armed PEG, wherein 7-8 anti-Factor D antibodies or antibody variants are covalently linked to the PEG.
在一些实施方案中，本发明的缀合物包含多臂PEG，所述多臂PEG具有通式(Ia)、(Ib)、(IIa)、(IIIa)或(IVa)中任一个的结构。在这种实施方案中，至少一个R2与本文描述的抗-因子D抗体或抗体变体共价连接。在一些实施方案中，具有通式(Ia)、(Ib)、(IIa)、(IIIa)或(IVa)中任一个的结构的多臂PEG是八聚体，并且至少两个、至少三个、至少四个、至少五个、至少六个、至少七个或全部八个R2基团与本文描述的抗-因子D抗体或抗体变体共价连接。In some embodiments, the conjugates of the invention comprise a multi-armed PEG having a structure of any of the general formulas (Ia), (Ib), (IIa), (Ilia), or (IVa). In such embodiments, at least oneR2 is covalently linked to an anti-Factor D antibody or antibody variant described herein. In some embodiments, the multi-armed PEG having a structure of any of formulas (Ia), (Ib), (IIa), (IIIa), or (IVa) is an octamer, and at least two, at least three , at least four, at least five, at least six, at least seven, or all eightR groups are covalently linked to an anti-Factor D antibody or antibody variant described herein.
在一些方面，本发明的缀合物包括这样的种类，其中多臂多元醇共价连接至亲本抗体上的一个或多个特定位点；即，聚合物连接靶向亲本抗体或抗体片段中的特定区域或一个或多个特定氨基酸残基。标准诱变技术可以用于改变亲本抗体或抗体片段中潜在的PEG化位点的数量和/或位置。因此，就氨基酸置换引入或替代氨基酸(诸如半胱氨酸和赖氨酸)的程度而言，本发明的抗-因子D抗体及其变体可以含有比天然序列抗-因子D更多或更少数量的潜在PEG化位点(如图1所示)。In some aspects, conjugates of the invention include species in which a multi-armed polyol is covalently linked to one or more specific sites on a parent antibody; A specific region or one or more specific amino acid residues. Standard mutagenesis techniques can be used to alter the number and/or location of potential PEGylation sites in the parental antibody or antibody fragment. Thus, to the extent that amino acid substitutions introduce or replace amino acids such as cysteine and lysine, the anti-Factor D antibodies of the invention and variants thereof may contain more or less than the native sequence anti-Factor D Small number of potential PEGylation sites (shown in Figure 1).
如上文所讨论的，聚合物的位点特异性缀合最常见地是通过与亲本抗体或抗体片段中的半胱氨酸残基连接来实现的。在这样的实施方案中，偶联化学可以例如利用不在亲本抗体的二硫桥键中的半胱氨酸残基的游离巯基。As discussed above, site-specific conjugation of polymers is most commonly achieved through attachment to cysteine residues in the parent antibody or antibody fragment. In such embodiments, the coupling chemistry may, for example, utilize free sulfhydryl groups of cysteine residues that are not in the disulfide bridges of the parent antibody.
在一些实施方案中，天然存在于亲本抗体中的一个或多个半胱氨酸残基被用作用于聚合物缀合的一个或多个连接位点。在其它实施方案中，抗体或抗体变体上的游离氨基可以用2-亚氨基-四氢噻吩(thiolane)(Traut’s试剂)硫醇化，并且然后与例如马来酰亚胺官能化的PEG偶联，如在Pedley等人,Br.J.Cancer,Vol.70,pp.1126-1130(1994)中描述的。在另一个实施方案中，将一个或多个半胱氨酸残基工程改造到亲本抗体中的所选择的一个或多个位点中以用于为聚合物提供一个或多个特定连接位点。In some embodiments, one or more cysteine residues naturally occurring in the parent antibody are used as one or more attachment sites for polymer conjugation. In other embodiments, free amino groups on the antibody or antibody variant can be thiolated with 2-imino-thiolane (Traut's reagent) and then coupled with, for example, maleimide functionalized PEG , as described in Pedley et al., Br. J. Cancer, Vol. 70, pp. 1126-1130 (1994). In another embodiment, one or more cysteine residues are engineered into one or more selected sites in the parent antibody for use in providing one or more specific attachment sites for the polymer .
先前已经描述了半胱氨酸工程改造的抗体(美国专利公开号2007/0092940和Junutula,J.R.等人,J.Immunol Methods,Vol.332(l-2),pp.41-52(2008)，全部通过引用整体结合于此)。在一些实施方案中，半胱氨酸工程改造的抗体可以是亲本抗体。这些可用于产生在特定位置(通常在恒定结构域，例如CL或CH1)具有游离半胱氨酸的抗体片段。经工程改造以含有半胱氨酸的亲本抗体在本文中称为“ThioMab”，并且由这种半胱氨酸工程改造的抗体制备的Fab片段(不论制备方法)在本文中称为“ThioFab”。如先前所描述的(参见例如美国专利公开号2007/0092940和Junutula,J.R.等人,J.Immunol Methods,Vol.332(l-2),pp.41-52(2008))，对于具有替代的(“工程改造的”)半胱氨酸(Cys)残基的突变体，评价新引入的工程改造的半胱氨酸硫醇基团的反应性。硫醇反应性值是范围为0至1.0的相对数值项，并且可以对任何半胱氨酸改造的抗体进行测量。除具有反应性硫醇基团之外，应该选择ThioMab以便它们保持抗原结合能力。先前详细描述了半胱氨酸工程改造的抗体的设计、选择和制备(参见例如WO 2011/069104，其通过引用结合于此)。在一些实施方案中，工程改造的半胱氨酸被引入到重链或轻链的恒定结构域中。同样地，半胱氨酸工程改造的抗体保持它们的野生型(亲本抗体对应物)的抗原结合能力，并且同样地能够与抗原特异性结合。Cysteine-engineered antibodies have been previously described (US Patent Publication No. 2007/0092940 and Junutula, JR et al., J. Immunol Methods, Vol. 332(1-2), pp. 41-52 (2008), All are hereby incorporated by reference in their entirety). In some embodiments, a cysteine engineered antibody can be a parental antibody. These can be used to generate antibody fragments with free cysteines at specific positions, usually in the constant domains, such asCL orCH1 . Parent antibodies engineered to contain cysteines are referred to herein as "ThioMabs" and Fab fragments (regardless of method of preparation) prepared from such cysteine-engineered antibodies are referred to herein as "ThioFabs" . As previously described (see e.g. US Patent Publication No. 2007/0092940 and Junutula, JR et al., J. Immunol Methods, Vol. 332(1-2), pp.41-52 (2008)), for ("Engineered") mutants of cysteine (Cys) residues were evaluated for reactivity of newly introduced engineered cysteine thiol groups. Thiol reactivity values are relative values ranging from 0 to 1.0 and can be measured for any cysteine engineered antibody. In addition to having reactive thiol groups, ThioMabs should be chosen so that they retain antigen binding ability. The design, selection and preparation of cysteine engineered antibodies have been previously described in detail (see eg WO 2011/069104, which is hereby incorporated by reference). In some embodiments, engineered cysteines are introduced into the constant domains of the heavy or light chains. Likewise, cysteine-engineered antibodies retain their wild-type (parental antibody counterpart) antigen-binding ability and are likewise capable of specifically binding antigen.
在一些方面，本发明涉及抗体片段-聚合物缀合物，其中所述抗体片段是Fab，并且所述聚合物连接至通常将形成连接轻链和重链的链间二硫键的Fab片段的轻链或重链中的一个或多个半胱氨酸残基。In some aspects, the invention relates to antibody fragment-polymer conjugates, wherein the antibody fragment is a Fab and the polymer is attached to the Fab fragment that would normally form an interchain disulfide bond linking the light and heavy chains. One or more cysteine residues in the light or heavy chain.
在另一个方面，本发明涉及抗体片段-聚合物缀合物，其中所述抗体片段是Fab-C，并且聚合物连接靶向Fab-C片段的铰链区。在一些实施方案中，天然存在于抗体片段的铰链区中的一个或多个半胱氨酸残基被用于连接聚合物。在另一个实施方案中，将一个或多个半胱氨酸残基工程改造到Fab-C片段的铰链区中以用于为聚合物提供一个或多个特定连接位点。在一些实施方案中，本文公开的抗-因子D抗体变体Fab片段通过在C’-末端添加一个半胱氨酸进行修饰，以为聚合物缀合提供一个连接位点。在另一个实施方案中，本文描述的抗-因子D抗体变体Fab片段通过在C’-末端添加四个另外的半胱氨酸Cys-Pro-Pro-Cys(SEQID NO:21)进行修饰，以为聚合物缀合提供两个连接位点。在又一个实施方案中，本文描述的抗-因子D抗体变体Fab片段通过在C’-末端添加四个另外的半胱氨酸Ser-Pro-Pro-Cys(SEQ ID NO:111进行修饰，以为聚合物缀合提供一个连接位点。在又一个实施方案中，本文描述的抗-因子D抗体变体Fab片段通过在C’-末端添加四个另外的半胱氨酸Cys-Pro-Pro-Ser(SEQ ID NO:112)进行修饰，以为聚合物缀合提供一个连接位点。在又一个实施方案中，本文描述的抗-因子D抗体变体Fab片段通过在C’-末端添加四个另外的半胱氨酸Ala-Pro-Pro-Cys(SEQ ID NO:113)进行修饰，以为聚合物缀合提供一个连接位点。在又一个实施方案中，本文描述的抗-因子D抗体变体Fab片段通过在C’-末端添加四个另外的半胱氨酸Ser-Gly-Gly-Cys(SEQ ID NO:114)进行修饰，以为聚合物缀合提供一个连接位点。在又一个实施方案中，本文描述的抗-因子D抗体变体Fab片段具有已经修饰为以“CYGPPC”结束的C’-末端，这为聚合物缀合提供一个连接位点。In another aspect, the invention relates to antibody fragment-polymer conjugates, wherein the antibody fragment is Fab-C and the polymer linkage is targeted to the hinge region of the Fab-C fragment. In some embodiments, one or more cysteine residues naturally occurring in the hinge region of the antibody fragment are used to attach the polymer. In another embodiment, one or more cysteine residues are engineered into the hinge region of the Fab-C fragment for providing one or more specific attachment sites for the polymer. In some embodiments, the anti-Factor D antibody variant Fab fragments disclosed herein are modified by the addition of a cysteine at the C'-terminus to provide an attachment site for polymer conjugation. In another embodiment, the anti-Factor D antibody variant Fab fragment described herein is modified by adding four additional cysteines Cys-Pro-Pro-Cys (SEQ ID NO: 21 ) at the C'-terminus, Provides two attachment sites for polymer conjugation. In yet another embodiment, the anti-Factor D antibody variant Fab fragments described herein are modified by adding four additional cysteines Ser-Pro-Pro-Cys (SEQ ID NO: 111, To provide a linking site for polymer conjugation. In yet another embodiment, the anti-Factor D antibody variant Fab fragments described herein are modified by adding four additional cysteines Cys-Pro-Pro at the C'-terminal -Ser (SEQ ID NO:112) is modified to provide a linking site for polymer conjugation. In yet another embodiment, the anti-Factor D antibody variant Fab fragments described herein are modified by adding four An additional cysteine Ala-Pro-Pro-Cys (SEQ ID NO: 113) is modified to provide a linking site for polymer conjugation. In yet another embodiment, the anti-Factor D antibody described herein The variant Fab fragment was modified by adding four additional cysteines Ser-Gly-Gly-Cys (SEQ ID NO: 114) at the C'-terminus to provide a linking site for polymer conjugation. In yet another In embodiments, the anti-Factor D antibody variant Fab fragments described herein have a C'-terminus that has been modified to end with "CYGPPC", which provides a linking site for polymer conjugation.
PEG化的程度和位点还可以通过调节反应条件来操纵，诸如官能化PEG和蛋白质的相对浓度，以及pH。所需PEG化程度的合适的条件可以通过改变标准PGE化反应的参数来经验地确定。The degree and site of PEGylation can also be manipulated by adjusting reaction conditions, such as the relative concentrations of functionalized PEG and protein, and pH. Suitable conditions for the desired degree of PEGylation can be determined empirically by varying the parameters of standard PGEylation reactions.
通过任何常规方法进行抗-因子D抗体和抗体变体的PEG化。合适的PEG化条件在WO2011/069104和WO 03/029420中陈述，这两者通过引用整体结合于此。PEGylation of anti-Factor D antibodies and antibody variants is performed by any conventional method. Suitable PEGylation conditions are set out in WO2011/069104 and WO 03/029420, both of which are hereby incorporated by reference in their entirety.
c.表征和活性c. Characterization and activity
PEG化蛋白质可以通过SDS-PAGE、凝胶过滤、NMR、肽作图(peptide mapping)、液相色谱-质谱分光光度法和体外生物学测定来表征。通常首先通过SDS-PAGE显示fab化的程度。10％SDS中的聚丙烯酰胺凝胶电泳通常在10mM Tris-HC1pH 8.0、100mM NaCl作为洗脱缓冲液中进行。为了证明哪种残基是PEG化的，可以使用蛋白酶(诸如胰蛋白酶和Lys-C蛋白酶)进行肽作图。因此，PEG化和非PEG化抗体的样品可以用蛋白酶(诸如Lys-C蛋白酶)消化，并且通过诸如反相HPLC的技术分离所获得的肽。所产生的肽的色谱图可以与先前针对抗-因子D多肽确定的肽图谱进行比较。PEGylated proteins can be characterized by SDS-PAGE, gel filtration, NMR, peptide mapping, liquid chromatography-mass spectrophotometry, and in vitro biological assays. The extent of fabization is usually first visualized by SDS-PAGE. Polyacrylamide gel electrophoresis in 10% SDS is usually performed in 10 mM Tris-HCl pH 8.0, 100 mM NaCl as elution buffer. To demonstrate which residues are PEGylated, peptide mapping can be performed using proteases such as trypsin and Lys-C protease. Thus, samples of PEGylated and non-PEGylated antibodies can be digested with a protease, such as Lys-C protease, and the resulting peptides separated by techniques such as reversed-phase HPLC. Chromatograms of the resulting peptides can be compared to peptide profiles previously determined for anti-Factor D polypeptides.
然后可以通过质谱分析每个峰来验证峰中缀合物的大小。取决于缀合中使用的PEG和在峰中缀合物的大小，可以估计缀合至PEG的抗体或其变体的数量。与PEG基团缀合的一个或多个片段通常在注射后不保留在HPLC柱上并从色谱中消失。这种从色谱中的消失表明该特定片段上的PEG化应该含有至少一个可PEG化的氨基酸残基。可以使用本领域已知的方法进一步测定PEG化的抗-因子D抗体和抗体变体与因子D相互作用的能力和其它生物活性。Each peak can then be analyzed by mass spectrometry to verify the size of the conjugate in the peak. Depending on the PEG used in conjugation and the size of the conjugate in the peak, the amount of antibody or variant thereof conjugated to PEG can be estimated. One or more fragments conjugated to a PEG group typically do not remain on the HPLC column after injection and disappear from the chromatography. This disappearance from the chromatogram indicates that PEGylation on this particular fragment should contain at least one PEGylatable amino acid residue. The ability of PEGylated anti-Factor D antibodies and antibody variants to interact with Factor D and other biological activities can be further assayed using methods known in the art.
PEG化改变抗体药物的物理和化学性质，并且可以导致改善的药物动力学表现，包括但不限于：改善的稳定性；降低的免疫原性；增加的流体动力学半径(RH)；和/或延长的循环寿命，以及增加的眼部停留时间。PEGylation alters the physical and chemical properties of the antibody drug and can lead to improved pharmacokinetic performance, including but not limited to: improved stability; reduced immunogenicity; increased hydrodynamic radius (RH); and/or Extended cycle life, and increased eye dwell time.
在一些实施方案中，与相应的未缀合的抗-因子D抗体或抗体变体相比，本发明的缀合物在经由单次玻璃体内注射施用至哺乳动物眼睛(例如人)后具有增加的半衰期。在一些实施方案中，半衰期的增加是相应的未缀合的抗-因子D抗体或抗体变体的半衰期的至少1.4倍、或至少1.5倍、或至少1.8倍、或至少2倍。In some embodiments, the conjugates of the invention have an increased factor D after administration to a mammalian eye (eg, a human) via a single intravitreal injection compared to a corresponding unconjugated anti-Factor D antibody or antibody variant. half-life. In some embodiments, the increase in half-life is at least 1.4-fold, or at least 1.5-fold, or at least 1.8-fold, or at least 2-fold the half-life of a corresponding unconjugated anti-Factor D antibody or antibody variant.
在一些实施方案中，本发明的缀合物可以具有以下的流体动力学半径(RH)：约3nm至约30nm或更大，或备选地约5nm至约25nm，并且在一些实施方案中可以是约5nm、约10nm、约15nm、约20nm、约25nm或更大，如使用本领域已知的方法确定的。In some embodiments, the conjugates of the invention may have a hydrodynamic radius (RH) of from about 3 nm to about 30 nm or greater, or alternatively from about 5 nm to about 25 nm, and in some embodiments may is about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, or greater, as determined using methods known in the art.
在一些实施方案中，本发明的缀合物可以展示的稳定性(通过使用本领域已知的方法测量的结合能力(例如，结合fD的能力)损失％/月来表征)(例如，当暴露于生理条件时)为约15％、约12％、约10％、约8％、或甚至更小。In some embodiments, the conjugates of the invention may exhibit stability (characterized by % loss per month of binding capacity (e.g., ability to bind fD) measured using methods known in the art) (e.g., when exposed to Under physiological conditions) is about 15%, about 12%, about 10%, about 8%, or even less.
在一些实施方案中，本发明的缀合物可以展示IC50效力值，如使用因子D依赖性因子B激活的时间分辨荧光能量转移(TR-FRET)测定所确定的，如实施例中详细描述的。在一些实施方案中，缀合物抑制因子D依赖性因子B激活，其中IC50值为约25pM至约10nM、或约25pM至约5nM、或约25pM至约1nM、或约25pM至约750pM、或约25pM至约500pM。In some embodiments, the conjugates of the invention can exhibit IC50 potency values as determined using a time-resolved fluorescence energy transfer (TR-FRET) assay for Factor D-dependent Factor B activation, as described in detail in the Examples . In some embodiments, the conjugate inhibits Factor D-dependent Factor B activation with anIC50 value of about 25 pM to about 10 nM, or about 25 pM to about 5 nM, or about 25 pM to about 1 nM, or about 25 pM to about 750 pM, Or about 25pM to about 500pM.
在一些实施方案中，缀合物具有使其适于通过窄孔径针施用的粘度。在一些实施方案中，在150-250mg/ml的浓度下，缀合物的粘度小于800cP，小于700cP，小于600cP，小于500cP，小于400cP，小于300cP，小于200cP，小于100cP，小于50cP，或小于30cP。在一些实施方案中，在200mg/ml的浓度下，缀合物的粘度小于300cP。In some embodiments, the conjugate has a viscosity that makes it suitable for administration through a narrow bore needle. In some embodiments, the viscosity of the conjugate is less than 800 cP, less than 700 cP, less than 600 cP, less than 500 cP, less than 400 cP, less than 300 cP, less than 200 cP, less than 100 cP, less than 50 cP, or less than 30cP. In some embodiments, the viscosity of the conjugate is less than 300 cP at a concentration of 200 mg/ml.
药物制剂pharmaceutical preparations
可以通过将具有所需纯度的缀合物与通常在本领域中使用的任选的“药用”载体、赋形剂或稳定剂(所有这些都被称为”赋形剂”)混合来制备本发明的缀合物的治疗制剂以作为冻干制剂或水溶液储存。例如，缓冲剂、稳定剂、防腐剂、等渗剂、非离子去垢剂、抗氧化剂和其它混合添加剂。(参见Remington’s Pharmaceutical Sciences,第16版,A.Osol编著(1980))。这些添加剂在所用的剂量和浓度对于接受体必须是无毒性的。Can be prepared by admixing a conjugate of the desired purity with optional "pharmaceutically acceptable" carriers, excipients or stabilizers (all of which are referred to as "excipients") commonly used in the art Therapeutic formulations of the conjugates of the invention are stored as lyophilized formulations or as aqueous solutions. For example, buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants and other compounding additives. (See Remington's Pharmaceutical Sciences, 16th Edition, edited by A. Osol (1980)). These additives must be nontoxic to recipients at the dosages and concentrations employed.
缓冲剂有助于将pH维持在接近生理条件的范围内。它们优选地以范围为约2mM至约50mM的浓度存在。适用于本发明的缓冲剂包括有机和无机酸及其盐，诸如柠檬酸盐缓冲剂(例如，柠檬酸单钠-柠檬酸二钠混合物、柠檬酸-柠檬酸三钠混合物、柠檬酸-柠檬酸单钠混合物等)、琥珀酸盐缓冲剂(例如，琥珀酸-琥珀酸单钠混合物、琥珀酸-氢氧化钠混合物、琥珀酸-琥珀酸二钠混合物等)、酒石酸盐缓冲剂(例如，酒石酸-酒石酸钠混合物、酒石酸-酒石酸钾混合物、酒石酸-氢氧化钠混合物等)、富马酸盐缓冲剂(例如，富马酸-富马酸单钠混合物等)、富马酸盐缓冲剂(例如，富马酸-富马酸单钠混合物、富马酸-富马酸二钠混合物、富马酸单钠-富马酸二钠混合物等)、葡萄糖酸盐缓冲剂(例如，葡萄糖酸-葡萄糖酸钠混合物、葡萄糖酸-氢氧化钠混合物、葡萄糖酸-葡萄糖酸钾混合物等)、草酸盐缓冲剂(例如，草酸-草酸钠混合物、草酸-氢氧化钠混合物、草酸-草酸钾混合物等)、乳酸盐缓冲剂(例如，乳酸-乳酸钠混合物、乳酸-氢氧化钠混合物、乳酸-乳酸钾混合物等)以及乙酸缓冲剂(例如，乙酸-乙酸钠混合物、乙酸-氢氧化钠混合物等)。另外，可以提及的有磷酸盐缓冲剂、组氨酸缓冲剂和三甲胺盐诸如Tris。Buffers help maintain the pH in a range close to physiological conditions. They are preferably present at concentrations ranging from about 2 mM to about 50 mM. Buffers suitable for use in the present invention include organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-citric acid monosodium mixture, etc.), succinate buffer (for example, succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffer (for example, tartaric acid - sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g. fumaric acid-monosodium fumarate mixtures, etc.), fumarate buffers (e.g. , fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffer (eg, gluconate-glucose sodium oxalate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate buffer (for example, oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.) , lactate buffers (for example, lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetic acid buffers (for example, acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). In addition, phosphate buffers, histidine buffers and trimethylamine salts such as Tris may be mentioned.
可以添加防腐剂以阻止微生物生长，并且其可以以范围为0.2％-1％(w/v)的量添加。适用于本发明的防腐剂包括苯酚、苄醇、间甲酚、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、十八烷基二甲基苄基氯化铵、苯扎卤铵(例如，苯扎氯铵、苯扎溴铵、苯扎碘铵)、六甲氯铵、对羟基苯甲酸烷基酯(诸如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯)、儿茶酚、间苯二酚、环己醇和3-戊醇。Preservatives may be added to prevent microbial growth and may be added in amounts ranging from 0.2% to 1% (w/v). Preservatives suitable for use in the present invention include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g. , benzalkonium chloride, benzalkonium bromide, benzalkonium iodide), hexamethylammonium chloride, alkyl parabens (such as methyl paraben or propyl paraben), catechol, m- Hydroquinone, cyclohexanol and 3-pentanol.
可以添加等渗剂(有时也被称为“稳定剂”)以保证本发明的液体组合物的等渗性，并且其包括多元糖醇，优选三元或更高元糖醇，诸如甘油、赤藓醇、阿糖醇、木糖醇、山梨糖醇和甘露糖醇。Isotonic agents (also sometimes referred to as "stabilizers") may be added to ensure the isotonicity of the liquid compositions of the present invention, and include polysaccharide alcohols, preferably trivalent or higher sugar alcohols, such as glycerin, erythritol, Britol, Arabitol, Xylitol, Sorbitol, and Mannitol.
稳定剂是指宽的赋形剂种类，其在功能上覆盖的范围可以是从填充剂到添加剂，所述添加剂使治疗剂溶解或者有助于防止变性或粘附到容器壁上。典型的稳定剂可以是多元糖醇(以上例举的)；氨基酸，诸如精氨酸、赖氨酸、甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、丙氨酸、鸟氨酸、L-亮氨酸、2-苯丙氨酸、谷氨酸、苏氨酸等；有机糖或糖醇，诸如乳糖、海藻糖、水苏糖、甘露糖醇、山梨糖醇、木糖醇、核糖醇、肌醇(myoinisitol)，半乳糖醇、甘油等，包括环醇诸如肌醇(inositol)；聚乙二醇；氨基酸聚合物；含硫还原剂，诸如尿素、谷胱甘肽、硫辛酸、硫代乙酸钠、硫代甘油、α-单硫代甘油和硫代硫酸钠；低分子量多肽(即<10残基)；蛋白质，诸如人血清白蛋白、牛血清白蛋白、明胶或免疫球蛋白；亲水聚合物，诸如聚乙烯吡咯烷酮；单糖，诸如木糖、甘露糖、果糖、葡萄糖；二糖，诸如乳糖、麦芽糖、蔗糖；和三糖，诸如棉子糖；多糖，诸如葡聚糖。稳定剂可以以范围为0.1至10,000重量/重量份的活性蛋白质存在。Stabilizers refer to a broad class of excipients that can functionally range from fillers to additives that dissolve therapeutic agents or help prevent denaturation or adhesion to container walls. Typical stabilizers may be polysaccharide alcohols (exemplified above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L - Leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribose Alcohols, myoinisitol, galactitol, glycerol, etc., including cyclic alcohols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, Sodium thioacetate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (ie, <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins ; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose; and trisaccharides such as raffinose; polysaccharides such as dextran . Stabilizers may be present in the range of 0.1 to 10,000 parts w/w active protein.
可以添加非离子表面活性剂或去垢剂(也称为“湿润剂”)以促进治疗剂的溶解以及保护治疗性蛋白质免于搅拌导致的聚集，其也允许制剂暴露于剪切表面应力而不导致蛋白质的变性。合适的非离子表面活性剂包括聚山梨醇酯(20、80等)、泊洛沙姆(184、188等)、多元醇、聚氧乙烯山梨糖醇酐单酯(-20、-80等)。非离子表面活性剂可以以约0.05mg/ml至约1.0mg/ml(优选约0.07mg/ml至约0.2mg/ml)的范围存在。Nonionic surfactants or detergents (also referred to as "wetting agents") can be added to facilitate dissolution of the therapeutic agent and to protect the therapeutic protein from aggregation induced by agitation, which also allows the formulation to be exposed to shear surface stress without lead to protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), polyol, polyoxyethylene sorbitan monoester ( -20, -80 etc.). Nonionic surfactants may be present in the range of about 0.05 mg/ml to about 1.0 mg/ml (preferably about 0.07 mg/ml to about 0.2 mg/ml).
另外的混合赋形剂包括填充剂(例如淀粉)、螯合剂(例如EDTA)、抗氧化剂(例如，抗坏血酸、甲硫氨酸、维生素E)和助溶剂。本文中的制剂也可以含有多于一种活性成分，所述活性成分是被治疗的特定适应证所需的。在一些实施方案中，活性成分具有彼此不产生不利影响的互补活性。例如，可能需要进一步提供免疫抑制剂。这种分子以对预期目的有效的量适当地存在于组合中。活性成分还可以包埋于例如分别通过凝聚技术或界面聚合制备的微胶囊(例如，羟甲基纤维素微胶囊或明胶微胶囊和聚(甲基丙烯酸甲酯)微胶囊)、胶质药物递送系统(例如，脂质体、白蛋白微球体、微乳液、纳米粒子和纳米胶囊)或巨乳液(macroemulsion)中。这种技术在Remington‘s Pharmaceutical Sciences,第16版,A.Osal编著(1980)中公开。Additional mixing excipients include fillers (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and co-solvents. The formulations herein may also contain more than one active ingredient as required for the particular indication being treated. In some embodiments, the active ingredients have complementary activities that do not adversely affect each other. For example, it may be desirable to further provide immunosuppressants. Such molecules are suitably present in combination in amounts effective for the intended purpose. Active ingredients can also be embedded in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules), colloidal drug delivery, prepared for example by coacervation techniques or interfacial polymerization, respectively. systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. This technique is disclosed in Remington's Pharmaceutical Sciences, 16th Edition, edited by A. Osal (1980).
用于体内施用的制剂必须是无菌的。例如，这可通过借助无菌滤膜的过滤而轻易地实现。可以制备持续释放制备物。持续释放制备物的合适实例包括含有抗体或其抗体变体或片段(例如抗原结合片段)的固体疏水性聚合物的半透性基质，所述基质处于成形制品(例如，薄膜或微胶囊)形式。缓释基质的实例包括聚酯、水凝胶(例如，聚(2-羟乙基-甲基丙烯酸)或聚(乙烯醇))、聚交酯(美国专利号3,773,919)、L-谷氨酸和L-谷氨酸乙酯的共聚物、不可降解的乙烯-乙酸乙烯酯、可降解的乳酸-乙醇酸共聚物诸如LUPRON DEPOTTM(可注射微球，其由乳酸-乙醇酸共聚物和乙酸亮丙瑞林组成)以及聚-D-(-)-3-羟基丁酸。聚合物诸如乙烯-乙酸乙烯酯和乳酸-乙醇酸能够释放分子超过100天，而某些水凝胶在更短的时间段内释放蛋白质。当被包封的抗体留在体内很长时间时，它们可能由于在37℃暴露于湿气而变性或聚集，从而导致生物活性丧失以及可能的免疫原性的改变。可以根据所涉及的机制设计用于稳定化的合理策略。例如，如果发现聚集机制是通过硫-二硫互换的分子间S--S键形成，则可以通过以下方法实现稳定化：修饰巯基残基、从酸性溶液中冷冻干燥、控制水分含量、使用合适的添加剂以及开发特异性聚合物基质组合物。Preparations for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filters. Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers comprising the antibody or antibody variants or fragments thereof (e.g., antigen-binding fragments) in the form of shaped articles (e.g., films or microcapsules) . Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylic acid) or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), L-glutamic acid Copolymers with ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic-glycolic acid copolymers such as LUPRON DEPOTTM (injectable microspheres made of lactic-glycolic acid copolymer and acetic acid Leuprolide) and poly-D-(-)-3-hydroxybutyrate. Polymers such as ethylene-vinyl acetate and lactic-glycolic acid are capable of releasing molecules for over 100 days, while certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for long periods of time, they may denature or aggregate due to exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies for stabilization can be devised depending on the mechanisms involved. For example, if the aggregation mechanism is found to be intermolecular S--S bond formation via sulfur-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using Suitable additives and development of specific polymer matrix compositions.
用于预防或治疗眼部疾病或病症的本发明的缀合物通常通过以下方式使用：眼部、眼内和/或玻璃体内注射，和/或巩膜旁注射，和/或筋膜下(subtenon)注射，和/或脉络膜上注射，和/或以滴眼液和/或软膏的形式局部施用。本发明的这种缀合物可以通过多种方法递送，例如作为允许化合物缓慢释放到玻璃体内的装置和/或贮藏物经玻璃体内递送，包括在参考文献诸如Intraocular Drug Delivery,Jaffe,Jaffe,Ashton,和Pearson,编辑,Taylor&Francis(2006年3月)中描述的那些。在一个实例中，装置可以呈微型泵和/或基质和/或被动扩散系统和/或包封的小室(cell)的形式，其在延长的时间段内释放化合物(Intraocular Drug Delivery,Jaffe,Jaffe,Ashton,和Pearson,编辑,Taylor&Francis(2006年3月)。也可以使用其它施用方法，其包括但不限于局部、肠胃外、皮下、腹膜内、肺内、鼻内和病变内施用。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内或皮下施用。The conjugates of the present invention for the prophylaxis or treatment of ocular diseases or disorders are generally administered by ocular, intraocular and/or intravitreal injection, and/or parascleral injection, and/or subtenon ) injection, and/or suprachoroidal injection, and/or topical application in the form of eye drops and/or ointment. Such conjugates of the invention can be delivered by a variety of methods, e.g. intravitreal delivery as a device and/or depot that allows slow release of the compound into the vitreous, as included in references such as Intraocular Drug Delivery, Jaffe, Jaffe, Ashton , and those described in Pearson, eds., Taylor & Francis (March 2006). In one example, the device may be in the form of a micropump and/or a matrix and/or a passive diffusion system and/or an encapsulated cell that releases a compound over an extended period of time (Intraocular Drug Delivery, Jaffe, Jaffe , Ashton, and Pearson, eds., Taylor & Francis (March 2006). Other methods of administration may also be used, including but not limited to topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intralesional administration. Parenteral Infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
可以通过本领域中已知的方法和使用本领域中已知的成分来制备用于眼部、眼内或玻璃体内施用的制剂。对有效治疗的主要要求是适当穿透通过眼睛。与其中可以局部递送药物的眼睛前部疾病不同，视网膜疾病需要更加具有位点特异性的方法。滴眼液和软膏很少穿透眼睛的后部，并且血眼屏障妨碍被系统性施用的药物穿透到眼组织中。因此，通常，所选择的用于药物递送以治疗视网膜疾病(诸如AMD和CNV)的方法是直接的玻璃体内注射。通常以一定间隔重复玻璃体内注射，所述间隔取决于患者的状态以及被递送的药物的性质和半衰期。对于眼内(例如玻璃体内)穿透，通常优选的是具有较小尺寸的分子。在一个实施方案中，使用窄孔径针进行玻璃体内施用。在一个实施方案中，窄孔径针是30、29、28、27、26、25、24、23或22号。Formulations for ophthalmic, intraocular or intravitreal administration may be prepared by methods and using ingredients known in the art. A major requirement for effective treatment is proper penetration through the eye. Unlike diseases of the front of the eye, where drugs can be delivered locally, retinal diseases require a more site-specific approach. Eye drops and ointments rarely penetrate the back of the eye, and the blood-ocular barrier prevents systemically administered drugs from penetrating into the ocular tissue. Therefore, in general, the method of choice for drug delivery to treat retinal diseases such as AMD and CNV is direct intravitreal injection. Intravitreal injections are typically repeated at intervals that depend on the state of the patient and the nature and half-life of the drug being delivered. For intraocular (eg, intravitreal) penetration, molecules with smaller sizes are generally preferred. In one embodiment, intravitreal administration is performed using a narrow bore needle. In one embodiment, the narrow bore needle is 30, 29, 28, 27, 26, 25, 24, 23 or 22 gauge.
可以通过通常用于评价眼内疾病的各种终点来测量补体相关性眼睛病症(诸如AMD或CNV)的治疗功效。例如，可以评价视力丧失。可以通过但不限于例如以下方法来评价视力丧失：通过从基线到所需时间点的最佳校正视敏度(BCVA)的平均改变来测量(例如，其中BCVA是基于糖尿病性视网膜病早期治疗研究(ETDRS)视力表并且在4米的测试距离进行评价)，测量相比于基线在所需时间点在视敏度方面失去少于15个字母的受试者的比例，测量相比于基线在所需时间点在视敏度方面得到多于或等于15个字母的受试者的比例，测量在所需时间点视敏度Snellen等效值为20/2000以下的受试者的比例，测量NEI视功能问卷，测量所需时间点的CNV的尺寸和CNV的渗漏量，例如通过荧光血管造影术等。可以进行眼部评价，例如，其包括但不限于例如进行眼部检查、测量眼内压、评价视敏度、测量裂隙灯压力、评价眼内炎症等。The efficacy of treatment of a complement-associated ocular disorder, such as AMD or CNV, can be measured by various endpoints commonly used to assess intraocular disease. For example, vision loss can be assessed. Visual loss can be assessed by, but not limited to, methods such as: measured by mean change in best corrected visual acuity (BCVA) from baseline to desired time point (e.g., where BCVA is based on the Diabetic Retinopathy Early Treatment Study (ETDRS) visual acuity chart and evaluated at a test distance of 4 meters), measuring the proportion of subjects who lost less than 15 letters in visual acuity at the desired time point compared to baseline, measured compared to baseline at Proportion of subjects achieving greater than or equal to 15 letters in visual acuity at desired time point, measured Proportion of subjects with visual acuity Snellen equivalent below 20/2000 at desired time point, measured NEI visual function questionnaire, measure CNV size and CNV leakage at desired time points, such as by fluorescein angiography, etc. Ocular evaluations can be performed, for example, which include, but are not limited to, for example, performing an eye examination, measuring intraocular pressure, evaluating visual acuity, measuring slit lamp pressure, evaluating intraocular inflammation, and the like.
将有效治疗特定病症或病况的抗体或其抗体变体的量将取决于病症或病况的性质，并且可以通过标准临床技术来确定。在可能的情况下，首先需要在体外测定剂量-反应曲线和本发明的药物组合物，然后在对人进行测试之前在有用的动物模型系统中进行测定。The amount of an antibody or antibody variant thereof that will be effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. Where possible, dose-response curves and pharmaceutical compositions of the invention need to be determined first in vitro and then in useful animal model systems prior to testing in humans.
在一些实施方案中，可以将本文描述的抗体和缀合物配制成抗体在制剂中的浓度为：至少50mg/mL、至少75mg/mL、至少100mg/mL、至少125mg/mL、至少150mg/mL、至少175mg/mL、至少200mg/mL、至少225mg/mL、至少250mg/mL、至少275mg/mL、至少300mg/mL、至少325mg/mL、至少350mg/mL、至少375mg/mL、至少400mg/mL、至少425mg/mL、至少450mg/mL、至少475mg/mL或至少500mg/mL。在一些实施方案中，抗体在制剂中的量为至少100mg/mL。在一些实施方案中，抗体在制剂中的量为至少200mg/mL。在一些实施方案中，抗体在制剂中的量为至少300mg/mL。在一些实施方案中，可以将本文描述的抗体和缀合物配制成抗体在制剂中的浓度为：约50mg/mL至约500mg/mL、约50mg/mL至约300mg/mL、约100mg/mL至约500mg/mL、约100mg/mL至约300mg/mL、约200mg/mL至约500mg/mL、约200mg/mL至约400mg/mL、约200mg/mL至约300mg/mL、或约250mg/mL至约375mg/mL。In some embodiments, the antibodies and conjugates described herein can be formulated such that the concentration of the antibody in the formulation is: at least 50 mg/mL, at least 75 mg/mL, at least 100 mg/mL, at least 125 mg/mL, at least 150 mg/mL , at least 175mg/mL, at least 200mg/mL, at least 225mg/mL, at least 250mg/mL, at least 275mg/mL, at least 300mg/mL, at least 325mg/mL, at least 350mg/mL, at least 375mg/mL, at least 400mg/mL , at least 425 mg/mL, at least 450 mg/mL, at least 475 mg/mL, or at least 500 mg/mL. In some embodiments, the amount of antibody in the formulation is at least 100 mg/mL. In some embodiments, the amount of antibody in the formulation is at least 200 mg/mL. In some embodiments, the amount of antibody in the formulation is at least 300 mg/mL. In some embodiments, the antibodies and conjugates described herein can be formulated such that the concentration of the antibody in the formulation is: about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 300 mg/mL, about 100 mg/mL to about 500 mg/mL, about 100 mg/mL to about 300 mg/mL, about 200 mg/mL to about 500 mg/mL, about 200 mg/mL to about 400 mg/mL, about 200 mg/mL to about 300 mg/mL, or about 250 mg/mL mL to about 375 mg/mL.
在一些实施方案中，通过皮下注射来施用包含治疗性多肽、抗体或其抗体变体或其片段(例如抗原结合片段)的缀合物的水溶液。在另一个实施方案中，通过玻璃体注射来施用包含治疗性多肽、抗体或其抗体变体或其片段(例如抗原结合片段)的缀合物的水溶液。每剂可以是范围为约0.3mg至约30mg/眼。In some embodiments, an aqueous solution of a conjugate comprising a therapeutic polypeptide, antibody or antibody variant or fragment thereof (eg, an antigen-binding fragment) is administered by subcutaneous injection. In another embodiment, an aqueous solution of a conjugate comprising a therapeutic polypeptide, antibody or antibody variant or fragment thereof (eg, an antigen-binding fragment) is administered by intravitreal injection. Each dose may range from about 0.3 mg to about 30 mg/eye.
用于皮下施用的给药时间表可以从每月一次到每天一次变化，这取决于许多临床因素，包括疾病类型、疾病的严重度以及受试者对治疗剂的敏感性。The dosing schedule for subcutaneous administration can vary from monthly to daily, depending on a number of clinical factors, including the type of disease, severity of the disease, and the sensitivity of the subject to the therapeutic agent.
制品或试剂盒Products or kits
本发明的另一个实施方案是含有可用于治疗、预防和/或诊断本发明的抗体或其变体或其片段(例如抗原结合片段)所靶向的病症的物质。例如，本发明涉及含有可用于治疗、预防和/或诊断补体相关性病症的物质的制品。所述制品包括容器和在所述容器上或与所述容器相结合的标签或包装插页。合适的容器包括，例如，瓶、小药瓶、注射器等。容器可以由多种材料诸如玻璃或塑料制成。所述容器容纳了可有效用于治疗、预防和/或诊断补体相关性病症的组合物并且可以具有无菌接入口(例如所述容器可以是静脉内输液袋或是具有可由皮下注射针头刺透的瓶塞的小药瓶)。组合物中的至少一种活性剂是本发明的抗-因子D抗体缀合物。所述标签或包装插页指示所述组合物可用于治疗、预防和/或诊断特定病症。Another embodiment of the invention is a substance comprising a substance useful for the treatment, prevention and/or diagnosis of a disorder targeted by an antibody or variant or fragment thereof (eg, an antigen-binding fragment) of the invention. For example, the invention relates to articles of manufacture containing substances useful in the treatment, prevention and/or diagnosis of complement-associated disorders. The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be made of a variety of materials such as glass or plastic. The container contains a composition effective for the treatment, prevention, and/or diagnosis of a complement-associated disorder and may have a sterile access port (e.g., the container may be an IV bag or have a hypodermic needle-penetrable vials with corks). At least one active agent in the composition is an anti-Factor D antibody conjugate of the invention. The label or package insert indicates that the composition is useful for the treatment, prevention and/or diagnosis of a particular condition.
包装插页是指通常包含在治疗产品的商品包装中的使用说明，其含有关于涉及此类治疗产品的使用的适应症、用法、剂量、施用、禁忌症和/或警告的信息。在一些实施方案中，所述标签或包装插页指示所述组合物用于治疗补体相关性病症，诸如例如之前列出的任何病症，包括眼部病症，例如年龄相关性黄斑变性(AMD)。所述标签或包装插页还将包含用于将抗体组合物施用至患者的使用说明。Package Insert means the instructions normally included in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. In some embodiments, the label or package insert indicates that the composition is used to treat a complement-associated disorder, such as, for example, any of the disorders previously listed, including ocular disorders, such as age-related macular degeneration (AMD). The label or package insert will also contain instructions for administering the antibody composition to a patient.
因此，制品还可以含有第二容器，其包含药用缓冲液，诸如注射用抑菌水(BWFI)、磷酸盐缓冲盐水、林格氏(Ringer)溶液和葡萄糖溶液。它可以进一步包括从商业和用户观点来看所需的其它材料，包括其它缓冲液、稀释剂、滤器、针和注射器。Accordingly, the article of manufacture may also contain a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
在另一个实施方案中，还提供这样的试剂盒，所述试剂盒可用于各种目的，例如，用于治疗、预防和/或诊断补体相关性病症，用于补体相关性溶血测定，用于从细胞纯化或免疫沉淀因子D多肽。对于因子D多肽的分离和纯化，所述试剂盒可以含有与小珠(例如，琼脂糖小珠)偶联的抗-因子D抗体。可以提供这样的试剂盒，所述试剂盒包含用于在体外(例如在ELISA或蛋白质印迹法中)检测和定量因子D多肽的抗体。与制品一样，所述试剂盒包括容器和在所述容器上或与所述容器相结合的标签或包装插页。所述容器盛有包含本发明的缀合物的组合物，所述缀合物包含至少一种抗-因子抗体。可以包括另外的容器，其含有例如稀释剂和缓冲液、对照抗体。所述标签或包装插页可以提供组合物的描述以及用于预期的体外或检测用途的使用说明。所述标签或包装插页可以提供用于向受试者施用(例如抗体或其抗体片段(例如抗原结合片段)的使用说明。In another embodiment, kits are also provided which can be used for various purposes, for example, for the treatment, prevention and/or diagnosis of complement-associated disorders, for complement-associated hemolysis assays, for Purification or immunoprecipitation of Factor D polypeptide from cells. For the isolation and purification of Factor D polypeptide, the kit can contain an anti-Factor D antibody coupled to beads (eg, sepharose beads). Kits may be provided comprising antibodies for detection and quantification of Factor D polypeptide in vitro (eg, in an ELISA or Western blot). As with an article of manufacture, the kit includes a container and a label or package insert on or associated with the container. The container contains a composition comprising a conjugate of the invention comprising at least one anti-factor antibody. Additional containers containing eg diluents and buffers, control antibodies may be included. The label or package insert can provide a description of the composition and instructions for its intended in vitro or assay use. The label or package insert can provide instructions for administering, eg, the antibody or antibody fragment thereof (eg, an antigen-binding fragment) to a subject.
治疗用途therapeutic use
本发明的缀合物可以用于治疗哺乳动物。在一些实施方案中，缀合物被施用于非人哺乳动物以例如用于获得临床前数据。示例性的待治疗的非人哺乳动物包括非人灵长类、狗、猫、啮齿动物和进行临床前研究的其它哺乳动物。所述哺乳动物可以是用于要用所述抗体治疗的疾病的建立的动物模型，或者可以用于研究目的抗体的毒性。在这些实施方案中的每一个中，可以在哺乳动物上进行剂量逐步增大研究。The conjugates of the invention can be used to treat mammals. In some embodiments, the conjugates are administered to a non-human mammal, eg, for obtaining preclinical data. Exemplary non-human mammals to be treated include non-human primates, dogs, cats, rodents and other mammals undergoing preclinical research. The mammal may be an established animal model for the disease to be treated with the antibody, or may be used to study the toxicity of the antibody of interest. In each of these embodiments, dose escalation studies can be performed in mammals.
可以通过任何合适的手段施用缀合物，包括肠胃外施用、皮下施用、腹膜内施用、肺内施用和鼻内施用以及如有需要用于局部免疫抑制治疗、病灶内施用。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内或皮下施用。另外，缀合物适合通过脉冲输注(pulseinfusion)使用，特别是用递减剂量的抗体或其抗体变体或其片段(例如抗原结合片段)。在一些实施方案中，给药可以通过注射(诸如静脉内或皮下注射)进行，这部分地取决于施用是否是短暂的或长期的。在一个实施方案中，使用窄孔径针进行玻璃体内施用。在一个实施方案中，窄孔径针是30、29、28、27、26、25、24、23或22号。在一个实施方案中，使用可植入端口递送系统施用给药。The conjugates may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal and, if necessary for local immunosuppressive therapy, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Additionally, the conjugate is suitable for use by pulse infusion, particularly with decreasing doses of the antibody or antibody variant or fragment thereof (eg, antigen-binding fragment). In some embodiments, administration can be by injection (such as intravenous or subcutaneous injection), depending in part on whether the administration is transient or chronic. In one embodiment, intravitreal administration is performed using a narrow bore needle. In one embodiment, the narrow bore needle is 30, 29, 28, 27, 26, 25, 24, 23 or 22 gauge. In one embodiment, the administration is administered using an implantable port delivery system.
为了预防或治疗疾病，缀合物的合适剂量将取决于所治疗的疾病的类型、疾病的严重度和进程，抗体是否出于预防目的或治疗目的而施用、先前的疗法、患者的临床史以及对抗体的反应和主治医师的判断。For the prophylaxis or treatment of disease, the appropriate dosage of the conjugate will depend on the type of disease being treated, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history, and Response to antibodies and judgment of the attending physician.
取决于疾病的类型和严重度，可以施用足够量的缀合物以向患者提供约1至约25mg/眼(即，约0.015mg/kg至约0.36mg/kg，假设治疗一只眼睛)，不论是例如通过一次或多次单独的施用还是通过连续输注。缀合物的典型的日剂量可以足以提供范围为约1至约20mg/眼或更多，或者约1至约15mg/眼或更多的抗体，这取决于以上提及的因素。对于数天或更长时间的重复施用，取决于病况，持续治疗直至所需的疾病症状的抑制出现。然而，其它剂量方案可以是有用的。该疗法的过程可以容易地通过常规技术和测定监测。示例性的给药方案在WO 94/04188中公开。Depending on the type and severity of the disease, a sufficient amount of the conjugate may be administered to provide the patient with about 1 to about 25 mg/eye (i.e., about 0.015 mg/kg to about 0.36 mg/kg, assuming treatment of one eye), Whether for example by one or more separate administrations or by continuous infusion. A typical daily dosage of the conjugate may be sufficient to provide antibody in the range of about 1 to about 20 mg/eye or more, or about 1 to about 15 mg/eye or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The course of this therapy can be easily monitored by conventional techniques and assays. Exemplary dosing regimens are disclosed in WO 94/04188.
缀合物组合物可以以与良好医学实践相一致的方式来配制、给药和施用。在这种情况下考虑的因素包括所治疗的具体病症、所治疗的具体哺乳动物、个体患者的临床状况、病症的病因、药剂的递送位点、施用方法、施用时间表以及医学从业者已知的其它因素。待施用的缀合物的“治疗有效量”将由这样的考虑因素决定，并且是预防、改善或治疗疾病或病症所需的最小量。所述缀合物不需要但是任选地与目前用于预防或治疗所讨论病症的一种或多种药剂进行配制。所述其它药剂的有效量取决于制剂中存在的抗体或其抗体变体或其片段(例如抗原结合片段)的量、病症或治疗的类型以及以上讨论的其它因素。这些通常以如前文所使用的相同剂量以及施用途径使用，或以迄今为止采用的剂量的约1％至99％使用。The conjugate compositions can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiology of the condition, the site of delivery of the agent, the method of administration, the schedule of administration, and what is known to the medical practitioner. other factors. A "therapeutically effective amount" of a conjugate to be administered will be determined by such considerations, and is the minimum amount required to prevent, ameliorate or treat a disease or condition. The conjugate need not, but is optionally, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody or antibody variant or fragment thereof (eg, antigen-binding fragment) present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as previously used, or at about 1% to 99% of the dosages hitherto employed.
识别因子D作为其靶标的本文公开的抗体和包含这些抗体的缀合物可以用于治疗个体中的补体介导的(补体相关性)病症。这些病症与过度或不受控的补体激活相关。其包括：心肺分流术(cardiopulmonary bypass operation)期间的补体激活；由急性心肌梗死、动脉瘤、卒中、出血性休克、挤压伤、多器官衰竭、低血容量性休克和肠缺血后的缺血-再灌注引起的补体激活。这些病症还可以包括以下疾病或病况：炎性病况，诸如严重烧伤、内毒素血症、脓毒性休克、成人呼吸窘迫综合征、血液透析、过敏性休克、严重哮喘、血管性水肿、克罗恩氏病(Crohn’s disease)、镰状细胞性贫血、链球菌感染后肾小球肾炎和胰腺炎。所述病况可以是不良药物反应、药物变态反应、IL-2诱导的血管渗漏综合征或放射摄影造影剂变态反应的结果。病症可以是系统性的。其还可以包括自身免疫疾病，诸如系统性红斑狼疮、重症肌无力、类风湿性关节炎、阿尔茨海默病(Alzheimer’s disease)和多发性硬化。补体激活还与移植排斥相关。近来，补体激活和眼病之间显示强的相关性，所述眼病诸如年龄相关性黄斑变性、糖尿病性视网膜病和其它缺血相关性视网膜病、脉络膜新血管形成(CNV)、葡萄膜炎、糖尿病性黄斑水肿、病理性近视、希佩尔-林道病、眼的组织胞浆菌病、视网膜中央静脉阻塞(CRVO)、角膜新血管形成和视网膜新血管形成。在一个实施方案中，补体相关性病症是补体相关性眼部病症。在一个实施方案中，补体相关性眼部病症选自由以下组成的组：年龄相关性黄斑变性(AMD)(包括干性和湿性(非渗出性和渗出性)形式)、脉络膜新血管形成(CNV)、葡萄膜炎、糖尿病性视网膜病、缺血相关性视网膜病、糖尿病性黄斑水肿、病理性近视、希佩尔-林道病、眼的组织胞浆菌病、视网膜中央静脉阻塞(CRVO)、角膜新血管形成和视网膜新血管形成。在一个实施方案中，补体相关性眼部病症选自中期干性形式AMD或地图状萎缩(GA)。Antibodies disclosed herein and conjugates comprising these antibodies that recognize Factor D as their target can be used to treat complement-mediated (complement-associated) disorders in an individual. These disorders are associated with excessive or uncontrolled complement activation. These include: complement activation during cardiopulmonary bypass operation; ischemia following acute myocardial infarction, aneurysm, stroke, hemorrhagic shock, crush injury, multiorgan failure, hypovolemic shock, and intestinal ischemia Complement activation by blood-reperfusion. These conditions may also include the following diseases or conditions: inflammatory conditions such as severe burns, endotoxemia, septic shock, adult respiratory distress syndrome, hemodialysis, anaphylactic shock, severe asthma, angioedema, Crohn's Crohn's disease, sickle cell anemia, poststreptococcal glomerulonephritis, and pancreatitis. The condition may be the result of an adverse drug reaction, drug allergy, IL-2-induced vascular leak syndrome, or radiographic contrast media allergy. The condition can be systemic. It can also include autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Alzheimer's disease and multiple sclerosis. Complement activation is also associated with transplant rejection. Recently, a strong correlation has been shown between complement activation and eye diseases such as age-related macular degeneration, diabetic retinopathy and other ischemia-related retinopathy, choroidal neovascularization (CNV), uveitis, diabetes macular edema, pathological myopia, Hipper-Lindau disease, histoplasmosis of the eye, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization. In one embodiment, the complement-associated disorder is a complement-associated ocular disorder. In one embodiment, the complement-associated ocular disorder is selected from the group consisting of age-related macular degeneration (AMD) (including dry and wet (non-exudative and exudative) forms), choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, diabetic macular edema, pathological myopia, Hipper-Lindau disease, histoplasmosis of the eye, central retinal vein occlusion (CRVO) ), corneal neovascularization and retinal neovascularization. In one embodiment, the complement-related ocular disorder is selected from intermediate dry form AMD or geographic atrophy (GA).
包含因子D拮抗剂的缀合物可以单独施用或与至少第二治疗化合物组合施用。缀合物和任何第二治疗化合物的施用可以同时进行，例如，作为单个组合物或者作为两种以上不同的组合物，使用相同或不同的施用途径。备选地或另外地，所述施用可以以任何顺序相继进行。在某些实施方案中，在两种以上组合物的施用之间可以存在数分钟至数日至数周至数月的间隔。例如，可以首先施用包含因子D拮抗剂的缀合物，然后施用第二治疗化合物。然而，还考虑第二治疗化合物与缀合物同时施用或在缀合物之前施用。在一个实例中，因子D拮抗剂是抗-因子D抗体。在另外的实例中，抗-因子D抗体是本文所述的抗-因子D抗体变体。在一些实施方案中，第二治疗化合物选自HTRA1拮抗剂、ANG2拮抗剂(诸如例如US20090304694 A1中公开的抗-ANG2抗体)、TIE2拮抗剂(诸如例如美国专利号6,376,653中公开的抗-TIE2抗体)、VEGF拮抗剂(诸如例如以下各项中公开的VEGF拮抗剂：2015年2月26日公布的美国专利号6,884,879，和WO98/45331(贝伐单抗(bevacizumab)和其它人源化抗-VEGF抗体)；WO2005/012359和WO2005/044853(G6或B20系列抗体(例如G6-31、B20-4.1)，以及第二补体组分拮抗剂。在一个实例中，第二治疗化合物是HTRA1拮抗剂、ANG2拮抗剂、TIE2拮抗剂、或VEGF拮抗剂。在另外的实例中，HTRA1拮抗剂是抗-HTRA1抗体。在另一个实施方案中，ANG2拮抗剂是抗-ANG2抗体。在另一个实施方案中，TIE2拮抗剂是抗-TIE2抗体。在一些实施方案中，VEGF拮抗剂选自VEGF捕获剂(诸如aflibercept和抗-VEGF抗体(诸如贝伐单抗或ranabizumab)。Conjugates comprising a Factor D antagonist can be administered alone or in combination with at least a second therapeutic compound. Administration of the conjugate and any second therapeutic compound can be performed simultaneously, eg, as a single composition or as two or more different compositions, using the same or different routes of administration. Alternatively or additionally, said administrations may be performed sequentially in any order. In certain embodiments, there may be an interval of minutes to days to weeks to months between the administration of two or more compositions. For example, the conjugate comprising the Factor D antagonist can be administered first, followed by the second therapeutic compound. However, administration of the second therapeutic compound concurrently with the conjugate or prior to the conjugate is also contemplated. In one example, the Factor D antagonist is an anti-Factor D antibody. In additional examples, the anti-Factor D antibody is an anti-Factor D antibody variant described herein. In some embodiments, the second therapeutic compound is selected from an HTRA1 antagonist, an ANG2 antagonist (such as, for example, an anti-ANG2 antibody disclosed in US20090304694 A1), a TIE2 antagonist (such as, for example, an anti-TIE2 antibody disclosed in US Patent No. 6,376,653 ), VEGF antagonists (such as, for example, those disclosed in U.S. Patent No. 6,884,879, published February 26, 2015, and WO98/45331 (bevacizumab and other humanized anti- VEGF antibody); WO2005/012359 and WO2005/044853 (G6 or B20 series antibodies (eg, G6-31, B20-4.1), and second complement component antagonists. In one example, the second therapeutic compound is an HTRA1 antagonist , ANG2 antagonist, TIE2 antagonist, or VEGF antagonist. In another example, the HTRA1 antagonist is an anti-HTRA1 antibody. In another embodiment, the ANG2 antagonist is an anti-ANG2 antibody. In another embodiment wherein the TIE2 antagonist is an anti-TIE2 antibody. In some embodiments, the VEGF antagonist is selected from VEGF traps such as aflibercept and anti-VEGF antibodies (such as bevacizumab or ranabizumab ).
适用于与如本文所公开的包含抗-因子D抗体的缀合物组合施用的其它治疗剂是经典或备选补体途径的各种成员的拮抗剂(补体抑制剂)。因此，本文公开的缀合物可以与C1、C2、C3、C4、C5、C6、C7、C8和C9补体组分中的一种或多种的拮抗剂组合施用。在一些实施方案中，本文公开的包含抗-因子D的缀合物与C2和/或C4和/或C5补体组分的拮抗剂(诸如抗-C2和/或抗-C4和/或抗-C5抗体)组合。这种抗体是本领域中已知的和/或是可商购的。抗-C5抗体eculizumab(Alexion,Cheshire,CT,USA)已被批准用于治疗阵发性夜间血红蛋白尿(Paroxysmal nocturnal hemoglobinuria)(PNH)和非典型溶血性尿毒症综合征(aHUS)。其它补体抑制剂在例如美国公开号20050036991A1中公开。因此，本文公开的包含抗-因子D抗体的缀合物可以与有效量的一种或多种补体抑制剂(包括但不限于抗-C2和抗-C5抗体)组合施用，任选地与至少一种另外的因子D拮抗剂/抗体组合施用。Other therapeutic agents suitable for administration in combination with the anti-Factor D antibody-containing conjugates as disclosed herein are antagonists of various members of the classical or alternative complement pathways (complement inhibitors). Accordingly, the conjugates disclosed herein may be administered in combination with antagonists of one or more of the C1 , C2, C3, C4, C5, C6, C7, C8, and C9 complement components. In some embodiments, conjugates disclosed herein comprising anti-Factor D and antagonists of C2 and/or C4 and/or C5 complement components (such as anti-C2 and/or anti-C4 and/or anti- C5 antibody) combination. Such antibodies are known in the art and/or are commercially available. The anti-C5 antibody eculizumab (Alexion, Cheshire, CT, USA) has been approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). Other complement inhibitors are disclosed, eg, in US Publication No. 20050036991A1. Accordingly, a conjugate comprising an anti-Factor D antibody disclosed herein may be administered in combination with an effective amount of one or more inhibitors of complement, including but not limited to anti-C2 and anti-C5 antibodies, optionally with at least An additional Factor D antagonist/antibody combination is administered.
在一些实施方案中，本发明中治疗患有补体介导的病症的人受试者中的补体介导的病症包括向所述受试者施用有效量的治疗化合物，诸如因子D拮抗剂或包含因子D拮抗剂的缀合物，并且还包括向所述受试者施用有效量的第二治疗化合物，所述第二治疗化合物为HTRA1拮抗剂，ANG2拮抗剂，TIE2拮抗剂，VEGF拮抗剂，或C1、C2、C3、C4、C5、C6、C7、C8和C9补体组分中一个或多个的拮抗剂。在一个实例中，因子D拮抗剂是抗-因子D抗体，并且缀合物包含一个或多个抗-因子D抗体。在另外的实例中，抗-因子D抗体是本文描述的抗-因子D抗体变体，并且缀合物包含一个或多个抗-因子D抗体变体。在一个实例中，HTRA拮抗剂是抗-HTRA1抗体。在另一个实例中，ANG2拮抗剂是抗-ANG2抗体。在另一个实例中，TIE2拮抗剂是抗-TIE2抗体。在另一个实例中，VEGF拮抗剂是抗-VEGF抗体。在另一个实施方案中，C2和/或C4和/或C5补体组分的拮抗剂是抗-C2和/或抗-C4和/或抗-C5抗体。在一个实例中，补体介导的病症是补体相关性眼部病症。在一个实例中，眼部病症是年龄相关性黄斑变性(AMD)，包括非渗出性(例如中期干性AMD或地图状萎缩(GA))和渗出性(例如湿性AMD(脉络膜新血管形成(CNV))AMD，糖尿病性视网膜病(DR)，眼内炎和葡萄膜炎。在一个实例中，补体相关性眼部病况是中期干性AMD。在一个实例中，补体相关性眼部病况是地图状萎缩。在一个实例中，补体相关性眼部病况是湿性AMD(脉络膜新血管形成(CNV))。In some embodiments, treating a complement-mediated disorder in a human subject suffering from a complement-mediated disorder according to the invention comprises administering to said subject an effective amount of a therapeutic compound, such as a Factor D antagonist or comprising a conjugate of a Factor D antagonist, and further comprising administering to said subject an effective amount of a second therapeutic compound that is an HTRA1 antagonist, an ANG2 antagonist, a TIE2 antagonist, a VEGF antagonist, Or an antagonist of one or more of the C1, C2, C3, C4, C5, C6, C7, C8 and C9 complement components. In one example, the Factor D antagonist is an anti-Factor D antibody and the conjugate comprises one or more anti-Factor D antibodies. In additional examples, the anti-Factor D antibody is an anti-Factor D antibody variant described herein, and the conjugate comprises one or more anti-Factor D antibody variants. In one example, the HTRA antagonist is an anti-HTRA1 antibody. In another example, the ANG2 antagonist is an anti-ANG2 antibody. In another example, the TIE2 antagonist is an anti-TIE2 antibody. In another example, the VEGF antagonist is an anti-VEGF antibody. In another embodiment, the antagonist of the C2 and/or C4 and/or C5 complement component is an anti-C2 and/or anti-C4 and/or anti-C5 antibody. In one example, the complement-mediated disorder is a complement-associated ocular disorder. In one example, the ocular disorder is age-related macular degeneration (AMD), including non-exudative (eg, intermediate dry AMD or geographic atrophy (GA)) and exudative (eg, wet AMD (choroidal neovascularization) (CNV)) AMD, diabetic retinopathy (DR), endophthalmitis, and uveitis. In one example, the complement-associated ocular condition is intermediate dry AMD. In one example, the complement-associated ocular condition is geographic atrophy. In one example, the complement-related ocular condition is wet AMD (choroidal neovascularization (CNV)).
本文中的组合施用包括使用分开的制剂或单个药物制剂的共同施用，以及以任何顺序的连续施用，其中两种(或全部)活性剂同时发挥其生物学活性通常需要一段时间。Combination administration herein includes co-administration using separate formulations or a single pharmaceutical formulation, as well as sequential administration in any order, where it usually takes a period of time for both (or all) active agents to exert their biological activity simultaneously.
实施例Example
以下实施例是以说明性而非限制性的方式提供的。除非另有说明，实施例中提到的市售试剂根据制造商的说明书使用。以下实施例以及整个说明书中以ATCC登录号识别的那些细胞的来源是美国典型培养物保藏中心(American Type Culture Collection，10801University Boulevard，Manassas，VA 20110-2209)。The following examples are offered by way of illustration and not limitation. Unless otherwise stated, commercially available reagents mentioned in the examples were used according to the manufacturer's instructions. The source of those cells identified by ATCC accession numbers in the following examples and throughout the specification is the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209).
实施例1：产生抗-因子D抗体变体Example 1: Generation of Anti-Factor D Antibody Variants
Lampalizumab是一种通过结合至因子D上的外部位点而有效抑制因子D和补体旁路途径的人源化抗-因子D Fab片段，其目前处于临床开发中以用于治疗地图状萎缩(GA)(干性AMD的晚期形式)。Lampalizumab(FCFD4515S；下文中“aFD”)是由214个残基的轻链(SEQ ID NO:1)和223个残基的重链(SEQ ID NO:2)组成的抗体Fab片段。Lampalizumab, a humanized anti-factor D Fab fragment that potently inhibits factor D and the alternative complement pathway by binding to an exosite on factor D, is currently in clinical development for the treatment of geographic atrophy (GA ) (advanced form of dry AMD). Lampalizumab (FCFD4515S; hereinafter "aFD") is an antibody Fab fragment consisting of a 214-residue light chain (SEQ ID NO: 1 ) and a 223-residue heavy chain (SEQ ID NO: 2).
虽然GA中的人II期临床试验的结果表明在每月玻璃体内注射aFD的情况下获得了治疗效果，但是存在使用更高的药物剂量以实现甚至更好的效力的动机。同时，较低的给药频率将给患者提供改善的便利性，具有降低感染率和增加临床效力的潜在益处，并且可以有助于治疗患有较早期形式的干性AMD的患者。Although the results of a human phase II clinical trial in GA indicated that a therapeutic effect was obtained with monthly intravitreal injections of aFD, there is an incentive to use higher drug doses in order to achieve even better efficacy. At the same time, less frequent dosing would provide improved convenience to patients, have the potential benefit of reducing infection rates and increasing clinical efficacy, and could help treat patients with earlier forms of dry AMD.
进行努力以进一步改善野生型aFD(WT)的物理和化学稳定性，尤其是在低pH条件下和/或在高浓度在中性pH下的稳定性。轻链上的天冬氨酸残基Asp-30和重链上的天冬氨酸残基Asp-62(图1A)被鉴定为倾向于异构化。Asp异构化涉及脱水形成环状的亚酰胺中间体(Asu)，其通常可以在pH<8下长期存在并且在离子交换色谱法(IEC)时作为基础峰被检测到。环状中间体的形成在较低的pH下被加速。环状中间体水解以形成Asp或Iso-Asp，产生与起始材料相同的电荷状态并因此是无法通过IEC检测的，以上过程在较高的pH下更快。Asp-62(根据Kabat编号的Asp-61)的异构化似乎不影响潜能因为在Fab:fD复合物的晶体结构中其不与因子D接触。Katschke等人(2012)J.Biol.Chem.287:12886。Asp-30与轻链残基Asp-32和Asp-92一起与因子D上的碱性残基进行静电接触。Asp-30的异构化相当快速并且推测其导致观察到的抗体潜能的损失。Asp残基32和92的异构化也可能影响fD结合但是已知的是速率非常慢。位置30处的环状亚酰胺的形成及其随后水解为异天冬氨酸可能通过扰乱静电相互作用而不利地影响抗原结合。分离的碱性部分上的抗原结合测量表明环状中间体形式是有完全活性的，与作为结合损失的原因的异-asp形成一致。Efforts were made to further improve the physical and chemical stability of wild-type aFD (WT), especially under low pH conditions and/or at high concentrations at neutral pH. Aspartate residues Asp-30 on the light chain and Asp-62 on the heavy chain (Figure 1A) were identified as prone to isomerization. Asp isomerization involves dehydration to form a cyclic imide intermediate (Asu), which can usually persist for a long time at pH<8 and is detected as a fundamental peak in ion exchange chromatography (IEC). The formation of cyclic intermediates is accelerated at lower pH. Hydrolysis of the cyclic intermediate to form Asp or Iso-Asp yields the same charge state as the starting material and is therefore undetectable by IEC, faster at higher pH. Isomerization of Asp-62 (Asp-61 according to Kabat numbering) does not appear to affect potency as it is not in contact with Factor D in the crystal structure of the Fab:fD complex. Katschke et al. (2012) J. Biol. Chem. 287:12886. Asp-30, along with light chain residues Asp-32 and Asp-92, make electrostatic contacts with basic residues on Factor D. Isomerization of Asp-30 is rather rapid and presumably leads to the observed loss of antibody potential. Isomerization of Asp residues 32 and 92 may also affect fD binding but is known to be very slow. The formation of the cyclic imide at position 30 and its subsequent hydrolysis to isoaspartic acid may adversely affect antigen binding by disturbing electrostatic interactions. Antigen binding measurements on the isolated basic moiety indicated that the cyclic intermediate form was fully active, consistent with iso-asp formation as the cause of the loss of binding.
重链上的Asn-103(根据Kabat编号的Asn-101)易于脱酰胺，该反应在中性pH下比在微酸性pH(6-7)下以更高的速率进行。脱酰胺可以根据IEC时酸性峰的出现来进行检测。Asn脱酰胺，与Asp异构化一样，经由环状Asu中间体进行。然而，因为由Asn形成Asu仅在较高的pH发生(其中Asu被水解以形成Asp或异-Asp)，所以通常仅检测到酸性峰。Asn-103的侧链与因子D残基Arg-172形成氢键。该位点处的脱酰胺或环状亚酰胺中间体Asu的形成对抗原结合的影响是未知的。Asn-103 on the heavy chain (Asn-101 according to Kabat numbering) is susceptible to deamidation, which proceeds at a higher rate at neutral pH than at slightly acidic pH (6-7). Deamidation can be detected by the appearance of an acidic peak in IEC. Asn deamidation, like Asp isomerization, proceeds via a cyclic Asu intermediate. However, since the formation of Asu from Asn occurs only at higher pH (where Asu is hydrolyzed to form Asp or iso-Asp), usually only acidic peaks are detected. The side chain of Asn-103 forms a hydrogen bond with Factor D residue Arg-172. The effect of deamidation at this site or the formation of the cyclic imide intermediate Asu on antigen binding is unknown.
aFD.WT的pI(7.1)比典型的人源化Fab(pI 8-9)低。CDR-L1的组成(图1A)导致VL结构域上的负电荷簇。这些特征可能影响分子的溶解性，尤其是在低pH和低离子强度。此外，在37℃，aFD.WT的高浓度制剂即使在中性pH和生理离子强度也可能倾向于以更快的速率形成非共价二聚体。The pi of aFD.WT (7.1 ) is lower than typical humanized Fabs (pi 8-9). The composition of CDR-L1 (Fig. 1A) results in clusters of negative charges on the VL domain. These characteristics may affect the solubility of molecules, especially at low pH and low ionic strength. Furthermore, at 37 °C, high concentration formulations of aFD.WT may tend to form non-covalent dimers at a faster rate even at neutral pH and physiological ionic strength.
制备aFD.WT的若干变体以用于提高稳定性。遵循随试剂盒提供的方案，通过使用(Agilent)诱变试剂盒进行定点诱变来引入点突变。合成载有所需的密码子改变的寡核苷酸引物。通过DNA测序鉴定和确定具有设计的改变的质粒。对于小规模的表达和纯化，将DNA转化到大肠杆菌菌株64B4中。将单菌落挑取到含有50μg/mL羧苄青霉素(carbenecillin)(培养基制备代码A3232)的5mL LB培养基(培养基制备编码A2008)中，并且在14mL培养管中在以200RPM进行振荡的情况下在Innova培养箱中在37℃培养过夜。将这些培养物用于接种在1L带挡板的摇瓶中的250mL的完全豆粕(soy crap)培养基(培养基制备编码A4564)(50μg/mL羧苄青霉素)中。将培养物在30℃在以200RPM进行振荡的情况下培养过夜，然后通过离心收集。将细胞沉淀用PopCulture介质(Invitrogen)裂解，并且在Gravitrap Protein G柱(GE Healthcare)上遵循制造商提供的方案纯化Fab。对于较大规模的Fab制备，将来自10L转化细胞发酵的细胞糊悬浮在提取缓冲液中并且使用微型流化仪将其匀浆化。通过免疫亲和层析在Protein G-Sepharose或κ-select上捕获Fab并且用低pH缓冲液洗脱。将低pH洗脱物调至pH 5并且进一步通过阳离子交换色谱在S-Sepharose柱上进行纯化。纯化的蛋白质的身份通过质谱确认并且将汇集的级分浓缩至约10mg/mL，并且经由透析交换到PBS缓冲剂(pH 7.3)(本文中也被称为“PBS”；8mM磷酸氢二钠(Na2HPO4)，2mM磷酸二氢钾(KH2PO4)，140mM NaCl，2.7mM KCl)中。Several variants of aFD.WT were prepared for improved stability. Following the protocol provided with the kit, by using (Agilent) mutagenesis kit for site-directed mutagenesis to introduce point mutations. Oligonucleotide primers carrying the desired codon changes were synthesized. Plasmids with designed changes were identified and confirmed by DNA sequencing. For small-scale expression and purification, the DNA was transformed into E. coli strain 64B4. Pick a single colony into 5 mL of LB medium (medium preparation code A2008) containing 50 μg/mL carbenecillin (medium preparation code A3232) and shake at 200 RPM in a 14 mL culture tube Incubate overnight at 37°C in an Innova incubator. These cultures were used to inoculate 250 mL of complete soy crap medium (media preparation code A4564) (50 μg/mL carbenicillin) in 1 L baffled shake flasks. Cultures were grown overnight at 30°C with shaking at 200 RPM, then harvested by centrifugation. Cell pellets were lysed with PopCulture medium (Invitrogen) and Fabs were purified on Gravitrap Protein G columns (GE Healthcare) following the manufacturer's protocol. For larger scale Fab preparation, cell paste from 10 L transformed cell fermentation was suspended in extraction buffer and homogenized using a microfluidizer. Fabs were captured by immunoaffinity chromatography on Protein G-Sepharose or K-select and eluted with low pH buffer. The low pH eluate was adjusted to pH 5 and further purified by cation exchange chromatography on an S-Sepharose column. The identity of the purified protein was confirmed by mass spectrometry and the pooled fractions were concentrated to approximately 10 mg/mL and exchanged via dialysis into PBS buffer (pH 7.3) (also referred to herein as "PBS"; 8 mM disodium phosphate ( Na2 HPO4 ), 2mM potassium dihydrogen phosphate (KH2PO4), 140mM NaCl, 2.7mM KCl).
实施例2：抗-因子D抗体变体的生物活性Example 2: Biological Activity of Anti-Factor D Antibody Variants
测试有希望的单个及组合突变体的因子D(fD)结合亲和力及抑制因子D活性的能力。Promising single and combinatorial mutants were tested for Factor D (fD) binding affinity and ability to inhibit Factor D activity.
a.通过表面等离子共振(SPR)测量的因子D结合亲和力a. Factor D binding affinity measured by surface plasmon resonance (SPR)
通过在T200仪器上进行表面等离子共振(SPR)测量来确定因子D结合固定的aFD.WT及其变体的动力学和结合常数KD。遵循制造商描述的方案，使用抗-huFab捕获试剂盒(GE healthcare目录号28-9583-25)将抗体Fab片段固定在S系列CM5传感器芯片上。由针对浓度以2倍增量从0.39nM变化至25nM的人因子D溶液的60μL等分试样的注射记录的传感图，计算结合的动力学。流速为30μL/min，运行缓冲液为HBS-P+，分析温度为25℃，采用实时参比室减除，并且跟踪因子D注射后的解离达10分钟。在减除对于注射运行缓冲液所观察到的传感图后，使用BiaEval软件v4.1(GE Healthcare)根据1:1模型对数据进行分析以提取动力学和亲和力常数。through the Surface plasmon resonance (SPR) measurements were performed on a T200 instrument to determine the kinetics and binding constantKD of Factor D binding to immobilized aFD.WT and its variants. Antibody Fab fragments were immobilized on the S-series CM5 sensor chip using the anti-huFab capture kit (GE healthcare cat. no. 28-9583-25) following the manufacturer's described protocol. The kinetics of binding were calculated from sensorgrams recorded for injections of 60 μL aliquots of human Factor D solution varying in concentration from 0.39 nM to 25 nM in 2-fold increments. The flow rate was 30 μL/min, the running buffer was HBS-P+, the analysis temperature was 25°C, real-time reference chamber subtraction was used, and the dissociation of Factor D after injection was followed for 10 minutes. After subtracting the sensorgrams observed for injection of running buffer, the data were analyzed according to the 1:1 model using BiaEval software v4.1 (GE Healthcare) to extract kinetic and affinity constants.
表1：突变在对因子D的亲和力方面的影响Table 1 : Effect of Mutations on Affinity for Factor D
基于在aFD.WT的轻链可变结构域(VL；SEQ ID NO:3)和重链可变结构域(VH；SEQID NO:4)中的位置来对突变体进行命名和编号。野生型残基的单字母代号之后是序列位置，之后是置换的氨基酸的单字母代号。通过冒号将相同结构域上的多个变化分开。Mutants were named and numbered based on their position in the light chain variable domain (VL; SEQ ID NO:3) and heavy chain variable domain (VH; SEQ ID NO:4) of aFD.WT. The one-letter designation of the wild-type residue is followed by the sequence position, followed by the one-letter designation of the substituted amino acid. Multiple changes on the same domain are separated by colons.
如表1中所示，aFD.WT对fD具有高的亲和力，其为可通过SPR技术测定的极限(～10pM KD)。CDR-L1中的天冬氨酸残基28、30和31可以单独地分别用Ser、Glu和Ser置换，而不明显影响对fD的亲和力(表1)。相反，用Ser替代CDR-L1Asp32导致fD-结合的明显损失，不论是单独地测试(AFD.v4)还是与D28S和D31S突变体(AFD.v5)组合地测试。确定将VL-D30E、D31S和VH-D62E组合的三重突变体(“TM”(AFD.v6))以及向TM(AFD.v6)添加VL-D92E的四重突变体(TM.D92E(AFD.v7))的fD亲和力与野生型分子相当。VH-D62E是在进行异构化而不明显影响fD-结合的位点处的置换；VL-Asp92是具有缓慢异构化速率的抗原接触残基。将VL-D28S、D30E、D31S和VH-D62E组合的四重突变体“SIESD”(AFD.v8)显示对fD的亲和力的小的(～2倍)损失。在SIESD(AFD.v8)的情况下，VL-D92E置换导致对fD的亲和力的进一步损失(参见AFD.v10(表1中的SIESD.D92E))。As shown in Table 1, aFD.WT has a high affinity for fD, which is at the limit of what can be determined by SPR technique (~10 pM KD). Aspartic acid residues 28, 30 and 31 in CDR-L1 could be replaced individually with Ser, Glu and Ser, respectively, without significantly affecting the affinity for fD (Table 1). In contrast, replacement of CDR-L1Asp32 by Ser resulted in a clear loss of fD-binding, whether tested alone (AFD.v4) or in combination with the D28S and D31S mutants (AFD.v5). A triple mutant ("TM"(AFD.v6)) combining VL-D30E, D31S and VH-D62E and a quadruple mutant (TM.D92E(AFD.v6) adding VL-D92E to TM(AFD.v6) were identified. The fD affinity of v7)) is comparable to the wild-type molecule. VH-D62E is a substitution at a site that undergoes isomerization without appreciably affecting fD-binding; VL-Asp92 is an antigen-contacting residue with a slow isomerization rate. The quadruple mutant "SIESD" (AFD.v8) combining VL-D28S, D30E, D31S and VH-D62E showed a small (~2-fold) loss of affinity for fD. In the case of SIESD (AFD.v8), the VL-D92E substitution resulted in a further loss of affinity for fD (see AFD.v10 (SIESD.D92E in Table 1)).
针对利用其它残基进行的置换对脱酰胺的潜在位点进行测试。在共晶结构中，VL-N34和VH-N52都与fD接触，但是这些位点在中性pH条件下都不显示显著的脱酰胺速率。这些位点处的Ser置换导致亲和力的损失(表1；AFD.v9和AFD.v11)。VH残基Asn-103接触fD并且在PBS中具有可测量的脱酰胺速率。在SIESD(AFD.v8)的情况中，将Asn-103置换成Asp或Ser导致对fD的亲和力的小的可接受的损失(参见AFD.v12(SIESD.N103D)和AFD.v14(SIESD.N103S))(表1)。用Gln置换Asn-103导致结合亲和力的较大下降(参见AFD.v13(SIESD.N103Q)(表1)。与SIESD(AFD.v8)类似，向五重突变体SIESD.N103S(AFD.v14)添加VL-D92E的SIESD.N103S.D92E(AFD.v15)进一步导致对fD亲和力的4倍下降。Potential sites for deamidation were tested for substitution with other residues. In the co-crystal structure, both VL-N34 and VH-N52 are in contact with fD, but neither of these sites show significant deamidation rates at neutral pH. Ser substitution at these sites resulted in a loss of affinity (Table 1; AFD.v9 and AFD.v11). VH residue Asn-103 contacts fD and has a measurable deamidation rate in PBS. In the case of SIESD (AFD.v8), substitution of Asn-103 by Asp or Ser resulted in a small acceptable loss of affinity for fD (see AFD.v12 (SIESD.N103D) and AFD.v14 (SIESD.N103S ))(Table 1). Replacement of Asn-103 with Gln resulted in a larger drop in binding affinity (see AFD.v13 (SIESD.N103Q) (Table 1). Similar to SIESD (AFD.v8), towards the pentafold mutant SIESD.N103S (AFD.v14) Addition of VL-D92E to SIESD.N103S.D92E (AFD.v15) further resulted in a 4-fold decrease in affinity for fD.
b.因子D抑制测定b. Factor D inhibition assay
使用备选途径(AP)溶血测定测试aFD.WT和变体抑制因子D诱导的补体激活的能力。使用兔红血球(Er)的AP溶血测定之前已被描述。Pangburn(1998),Methods.Enzymol.162:639；Katschke等人(2009)J.Biol.Chem.284:10473。将Er(ColoradoSerum)用佛罗那(veronal)缓冲液(GVB)中的0.5％牛皮明胶洗涤三次并重悬。以2X浓度制备aFD的稀释物并将其添加到96孔聚丙烯板中。将Er悬浮液与GVB/0.1M EGTA/0.1M MgCl2混合并添加到板中。通过添加C1q耗尽的人血清来启动补体激活，以避免经由经典途径的任何补体激活(CompTech；1:3稀释在GVB中)。室温下温育30分钟后，通过添加GVB中的10mMEDTA来终止反应。将所述板离心并将上清液转移。在412nm处读取上清液的吸光度。通过非线性回归分析确定引起半最大抑制(IC50)的AFD.Ab浓度。The ability of aFD.WT and variants to inhibit factor D-induced complement activation was tested using an alternative pathway (AP) hemolytic assay. The AP hemolysis assay using rabbit erythrocytes (Er) has been described previously. Pangburn (1998), Methods. Enzymol. 162:639; Katschke et al. (2009) J. Biol. Chem. 284:10473. Er (ColoradoSerum) was washed three times with 0.5% kraft gelatin in veronal buffer (GVB) and resuspended. Dilutions of aFD were prepared at 2X concentration and added to 96-well polypropylene plates. The Er suspension was mixed with GVB/0.1M EGTA/0.1MMgCl2 and added to the plate. Complement activation was initiated by addition of C1q-depleted human serum to avoid any complement activation via the classical pathway (CompTech; 1:3 dilution in GVB). After 30 minutes of incubation at room temperature, the reaction was stopped by adding 10 mM EDTA in GVB. The plate was centrifuged and the supernatant transferred. The absorbance of the supernatant was read at 412 nm. The concentration of AFD.Ab that elicited half-maximal inhibition (IC50) was determined by non-linear regression analysis.
表2：AFD.Ab变体的抑制效力Table 2: Inhibition potency of AFD.Ab variants
*RSE＝±30％*RSE＝±30％
如表2中所示，变体SIESD(AFD.v8)、SIESD.N103S(AFD.v14)和TM.D92E(AFD.v7)具有抑制与aFD WT相当的fD依赖性补体激活活性的潜力，IC50测量中的标准差为±30％。As shown in Table 2, variants SIESD (AFD.v8), SIESD.N103S (AFD.v14) and TM.D92E (AFD.v7) have the potential to inhibit fD-dependent complement activation activity comparable to aFD WT with IC50 The standard deviation in the measurements is ±30%.
c.在延长的时间内的结合能力c. Ability to bind over an extended period of time
还使用SPR来测量在限定的条件下AFD.Ab变体与fD的随时间的总的结合。这些测量中的标准差为±10％。图2A显示在pH 5.5，对于aFD.WT和AFD.Ab变体D30E(AFD.v2)和TM(AFD.v6)，在一个月内，结合的损失约为40％，而对于SIESD(AFD.v8)和SIESD.N103S(AFD.v14)，即使在延长的时间内(70天)，结合的损失也较小，为约15％。作为比较，抗-VEGF抗体Fab片段(aVEGF)在70天内未显示结合的损失。对于aFD.WT和aVEGF，向pH 7.4条件添加盐似乎加快结合损失的速率(数据未显示)。如图2B中所示，在存在PBS的情况下(Fab蛋白浓度为100mg/ml)，D30E(AFD.v2)和TM(AFD.v6)具有相当的结合损失速率，其慢于对于aFD.WT所观察到的。在37℃ 10周后的结合损失为约30％(对于aFD.WT)和20％(对于抗-因子D变体D30E(AFD.v2)和TM(AFD.v6))。对于SIESD.N103S，在37℃ 10周后的结合损失仅为10％(AFD.14；图2B)，不大于实验误差，并且与在相同条件下对于aVEGF所观察到的相当。对SIESD(AFD.v8)，重复在PBS实验中在100mg/mL Fab浓度的热应激以收集达70天的数据。如图2C中所示，对于SIESD(AFD.v8)，70天时的结合损失小于10％。SPR was also used to measure the total binding of AFD.Ab variants to fD over time under defined conditions. The standard deviation in these measurements is ±10%. Figure 2A shows that at pH 5.5, for aFD.WT and AFD.Ab variants D30E (AFD.v2) and TM (AFD.v6), within one month, the loss of binding was about 40%, while for SIESD (AFD. v8) and SIESD.N103S (AFD.v14), even over an extended period of time (70 days), the loss of binding was small, about 15%. In comparison, the anti-VEGF antibody Fab fragment (aVEGF) showed no loss of binding over 70 days. Addition of salt to the pH 7.4 condition appeared to accelerate the rate of binding loss for aFD.WT and aVEGF (data not shown). As shown in Figure 2B, in the presence of PBS (Fab protein concentration of 100 mg/ml), D30E (AFD.v2) and TM (AFD.v6) had comparable binding loss rates, which were slower than for aFD.WT observed. Binding losses after 10 weeks at 37°C were approximately 30% (for aFD.WT) and 20% (for anti-factor D variants D30E (AFD.v2) and TM (AFD.v6)). For SIESD.N103S, the loss of binding after 10 weeks at 37°C was only 10% (AFD.14; Figure 2B), not greater than experimental error, and comparable to that observed for aVEGF under the same conditions. For SIESD (AFD.v8), heat stress at 100 mg/mL Fab concentration in PBS experiments was repeated to collect data for up to 70 days. As shown in Figure 2C, for SIESD (AFD.v8), the loss of binding at 70 days was less than 10%.
实施例3.具有改善的稳定性的抗-因子D抗体变体Example 3. Anti-Factor D Antibody Variants with Improved Stability
基于以上亲和力测定，选择若干单个或组合抗-因子D抗体变体用于进一步的稳定性分析。Based on the above affinity determinations, several single or combined anti-Factor D antibody variants were selected for further stability analysis.
a.溶解性a.Solubility
首先测试样品在低离子强度和pH 6的溶解性。首先通过使用Amicon CentriprepYM-10离心过滤单元浓缩至～100mg/mL而在20mM His-HCl pH 5缓冲液中制备样品。在肉眼观察时，这些溶液在pH 5和低离子强度未显示混浊。将样品以14,000xg离心10分钟以使任何不溶性物质沉淀。没有观察到沉淀，并且通过UV吸光度测量确定溶液的蛋白浓度。将样品(～1mL)置于10K MWCO的Slide-A-Lyzer盒(Pierce)中并在4℃对1L的20mM His缓冲液，pH6进行透析过夜，之后肉眼观察浊度。拍摄溶液的照片并提供在图6中。在pH 6和低离子强度条件(～100mg/ml，在20mM His-HCl，pH 6中)，aFD.WT和D30E(AFD.v2)溶液明显是混浊的，TM(AFD.v6)溶液没那么混浊，而SIESD(AFD.v8)的溶液是清的(图6)。在如以上那样离心后，对于aFD.WT和AFD.v2，肉眼观察到大的沉淀，对于TM(AFD.v6)，肉眼观察到大的沉淀，而对于SIESD(AFD.v8)，肉眼没有观察到沉淀，通过UV吸光度测量确定上清液的蛋白浓度(表3)。aFD.WT和D30E(AFD.v2)显示小于50mg/ml的溶解性，TM(AFD.v6)显示接近100mg/mL的溶解性而SIESD(AFD.v8)在这些条件下可全溶。对于SIESD(AFD.v8)，在pH 6透析后，蛋白浓度相对于pH 5起始浓度的小幅减小反映了透析后的稀释作用而不是AFD.v8的沉淀，因为在离心后未观察到沉淀。The samples were first tested for solubility at low ionic strength and pH 6. Samples were first prepared in 20 mM His-HCl pH 5 buffer by concentrating to ~100 mg/mL using an Amicon Centriprep YM-10 centrifugal filter unit. These solutions showed no turbidity at pH 5 and low ionic strength when observed with the naked eye. Samples were centrifuged at 14,000 xg for 10 minutes to pellet any insoluble material. No precipitation was observed, and the protein concentration of the solution was determined by UV absorbance measurement. Samples (-1 mL) were placed in a 10K MWCO Slide-A-Lyzer cassette (Pierce) and dialyzed overnight at 4°C against 1 L of 20 mM His buffer, pH 6, after which turbidity was visually observed. A photograph of the solution was taken and provided in Figure 6. At pH 6 and low ionic strength conditions (~100 mg/ml in 20 mM His-HCl, pH 6), aFD.WT and D30E (AFD.v2) solutions were visibly turbid, TM (AFD.v6) solutions were less so Cloudy, while the solution of SIESD (AFD.v8) was clear (Figure 6). After centrifugation as above, a large precipitate was visually observed for aFD.WT and AFD.v2, for TM (AFD.v6), and not for SIESD (AFD.v8) After precipitation, the protein concentration of the supernatant was determined by UV absorbance measurement (Table 3). aFD.WT and D30E (AFD.v2) showed a solubility of less than 50 mg/ml, TM (AFD.v6) showed a solubility of nearly 100 mg/mL and SIESD (AFD.v8) was fully soluble under these conditions. For SIESD (AFD.v8), the small decrease in protein concentration after dialysis at pH 6 relative to the starting concentration at pH 5 reflects the effect of dilution after dialysis rather than precipitation of AFD.v8, as no precipitation was observed after centrifugation .
表3.AFD.Ab变体的溶解性(～100mg/ml，在20mM His-HCl，pH 6中)Table 3. Solubility of AFD.Ab variants (-100 mg/ml in 20 mM His-HCl, pH 6)
在无盐溶解性测试中测试另外的变体AFD.v3、AFD.v12、AFD.v13和AFD.v14。在4℃透析到pH 6缓冲液中并在37℃孵育过夜后，除了aFD.WT以往的所有蛋白质溶液都是清的(图7)。37℃孵育和离心后对蛋白浓度的测量(表4)显示，所有变体的可溶性都比aFD.WT更好。在随后对PBS(pH7.3)，一种含有盐(NaCl)的缓冲液透析时，aFD.WT的混浊溶液(图7，顶行)变清，这暗示在添加盐和/或升高pH的情况下，沉淀是可逆的(图7，底行)。关于AFD.v3的溶解性数据指示，单个氨基酸变化D31S(除去1个带负电荷的残基)可以导致增加的溶解性。AFD.v8、AFD.v12、AFD.v13和AFD.v14中进一步的氨基酸变化也导致增加的溶解性。Additional variants AFD.v3, AFD.v12, AFD.v13 and AFD.v14 were tested in the no-salt solubility test. After dialysis at 4°C into pH 6 buffer and overnight incubation at 37°C, all previous protein solutions were clear except for aFD.WT (Figure 7). Measurement of protein concentration after incubation and centrifugation at 37°C (Table 4) showed that all variants were more soluble than aFD.WT. Upon subsequent dialysis against PBS (pH 7.3), a buffer containing salt (NaCl), the cloudy solution of aFD.WT (Fig. In the case of , the precipitation was reversible (Fig. 7, bottom row). Solubility data for AFD.v3 indicated that a single amino acid change D31S (removal of 1 negatively charged residue) could result in increased solubility. Further amino acid changes in AFD.v8, AFD.v12, AFD.v13 and AFD.v14 also resulted in increased solubility.
表4.AFD.Ab变体在pH 6、无盐情况下的溶解性Table 4. Solubility of AFD.Ab variants at pH 6 without salt
还测试了aFD.WT、SIESD(AFD.v8)和SIESD.N103S(AFD.v14)在生理pH(pH 7.3)和离子强度的条件下的溶解性。对于在生理pH和离子强度下的溶解性测试，将样品对PBS过夜透析，然后使用Amicon Centriprep YM-10离心过滤单元浓缩至227-372mg/mL。在4℃过夜孵育后，肉眼观察样品的浊度，将一部分浓缩以除去沉淀的蛋白质并通过UV吸光度测量确定蛋白浓度，并在表5中进行报告。在离心之前，aFD.WT样品是混浊的，而SIESD(AFD.v8)和SIESD.N103S(AFD.v14)的溶液是清的(图8中显示的aFD.WT、AFD.v8和AFD.v14)。对于照片(图8)中的溶液，AFD.v14的浓度为344mg/mL，然后其被进一步浓缩至372mg/mL。对于图8中的溶液，AFD.v8的浓度为269mg/ml。对于图8中的溶液，aFD.WT的浓度为227mg/mL。在离心后，在aFD.WT溶液的情况下观察到沉淀，但是对于SIESD(AFD.v8)和SIESD.N103S(AFD.v14)的溶液则没有观察到沉淀。蛋白浓度数据(表5)指示，在PBS中，aFD.WT仅能够被浓缩至227mg/mL，之后则观察到沉淀，而SIESD(AFD.v8)(≥269mg/mL)和SIESD.N103S(AFD.v14)(≥372mg/mL)的溶解性极限更高。因为SIESD(AFD.v8)在269mg/mL没有观察到沉淀，并且没有进行进一步浓缩溶液的尝试，所以这是该变体在PBS中的溶解性的下限。类似地，SIESD.N103S(AFD.v14)在PBS中的溶解性的下限是372mg/mL。在PBS中的SIESD(AFD.v8)的269mg/mL溶液在2-8℃孵育4周后保持清澈。类似地，在PBS中的SIESD.N103S(AFD.v14)的372mg/mL溶液在2-8℃孵育3周后浊度没有任何明显增加。在该浓度，在使用分子排阻色谱法(SEC)测量时，聚集体％的变化很小(图9)，在2-8℃在3周内，从0.9％增加至2.1％(3周孵育前的SEC数据(0.9％聚集体)显示在图9中；3周孵育后的SEC数据是未显示的数据)。The solubility of aFD.WT, SIESD (AFD.v8) and SIESD.N103S (AFD.v14) was also tested under conditions of physiological pH (pH 7.3) and ionic strength. For solubility testing at physiological pH and ionic strength, samples were dialyzed against PBS overnight and then concentrated to 227-372 mg/mL using an Amicon Centriprep YM-10 centrifugal filter unit. After overnight incubation at 4 °C, samples were visually inspected for turbidity, a portion was concentrated to remove precipitated protein and protein concentration was determined by UV absorbance measurement and reported in Table 5. Before centrifugation, aFD.WT samples were turbid, while solutions of SIESD (AFD.v8) and SIESD.N103S (AFD.v14) were clear (aFD.WT, AFD.v8, and AFD.v14 shown in Figure 8 ). For the solution in the photo (Figure 8), the concentration of AFD.v14 was 344 mg/mL, which was then further concentrated to 372 mg/mL. For the solution in Figure 8, the concentration of AFD.v8 was 269 mg/ml. For the solution in Figure 8, the concentration of aFD.WT was 227 mg/mL. After centrifugation, precipitation was observed in the case of the aFD.WT solution, but not for the solutions of SIESD (AFD.v8) and SIESD.N103S (AFD.v14). Protein concentration data (Table 5) indicated that aFD.WT could only be concentrated to 227 mg/mL in PBS, after which precipitation was observed, while SIESD (AFD.v8) (≥269 mg/mL) and SIESD.N103S (AFD .v14) (≥372mg/mL) has a higher solubility limit. Since no precipitation was observed for SIESD (AFD.v8) at 269 mg/mL and no further attempts were made to concentrate the solution, this was the lower limit of the solubility of this variant in PBS. Similarly, the lower limit of solubility of SIESD.N103S (AFD.v14) in PBS was 372 mg/mL. A 269 mg/mL solution of SIESD (AFD.v8) in PBS remained clear after incubation at 2-8°C for 4 weeks. Similarly, a 372 mg/mL solution of SIESD.N103S (AFD.v14) in PBS did not have any significant increase in turbidity after incubation at 2-8°C for 3 weeks. At this concentration, there was little change in % aggregates when measured using size exclusion chromatography (SEC) (Fig. 9), increasing from 0.9% to 2.1% within 3 weeks at 2-8°C (3 weeks incubation SEC data before (0.9% aggregates) are shown in Figure 9; SEC data after 3 weeks of incubation are data not shown).
表5.AFD.Ab变体的溶解性(在PBS(pH 7.3)中)Table 5. Solubility of AFD.Ab variants (in PBS (pH 7.3))
*pI值通过成像毛细管等电聚焦(icIEF)确定*pI values determined by imaging capillary isoelectric focusing (icIEF)
还比较了变体SIESD(AFD.v8)和SIESD.N103S(AFD.v14)在可以代表用于经由玻璃体内注射施用的药物的制剂的具有pH 5.5(20mM HCl pH 5.5)和不同NaCl浓度的缓冲液中的溶解性。制备蛋白浓度为约100mg/mL的溶液并且将其对测试缓冲液透析。然后使用Amicon Centriprep YM-10离心过滤单元将这些溶液浓缩。表6中列出了使溶液在环境温度保持在视觉上清澈的获得的浓度。SIESD(AFD.v8)在pH 5.5和低NaCl浓度具有达到314mg/mL的高溶解性。在添加100mM NaCl的情况下，SIESD.N103S(AFD.v14)的达到278mg/mL的高浓度也是可实现的。The variants SIESD (AFD.v8) and SIESD.N103S (AFD.v14) were also compared in buffers with pH 5.5 (20 mM HCl pH 5.5) and different NaCl concentrations, which may represent formulations of drugs administered via intravitreal injection. Solubility in liquid. Solutions with a protein concentration of approximately 100 mg/mL were prepared and dialyzed against assay buffer. These solutions were then concentrated using Amicon Centriprep YM-10 centrifugal filter units. Table 6 lists the concentrations obtained to keep the solutions visually clear at ambient temperature. SIESD (AFD.v8) has a high solubility reaching 314 mg/mL at pH 5.5 and low NaCl concentration. High concentrations of SIESD.N103S (AFD.v14) up to 278 mg/mL were also achievable with the addition of 100 mM NaCl.
表6.AFD.Ab变体在pH 5.5(20mM His-HCl pH 5.5)和不同NaCl浓度下的溶解性Table 6. Solubility of AFD.Ab variants at pH 5.5 (20 mM His-HCl pH 5.5) and different NaCl concentrations
NT＝未测NT = not tested
虽然与aFD.WT相比，SIESD.N103S(AFD.v14)具有两个(2)带较少负电的残基，但电荷的这些变化并未导致pI的明显变化(表5)，如通过成像毛细管等电聚焦测量的(iCIEF)(Salas-Solano等,J.Sep Sci,35(22):3124(2012))。预期蛋白质在接近pI的pH具有最小的溶解性(Green,A.A.,J.Biol.Chem.,93:517-542(1931))。对于SIESD.N103S(AFD.v14)，在PBS(pH 7.3)中的增加的溶解性与pI的改变不相关。更确切地，LC-CDR-L1中Asp到Ser的氨基酸变化(VL-D28S和D31S)表现为改变分子表面的电荷分布。Although SIESD.N103S (AFD.v14) has two (2) less negatively charged residues compared to aFD.WT, these changes in charge did not result in appreciable changes in pI (Table 5), as demonstrated by imaging Capillary isoelectric focusing measurements (iCIEF) (Salas-Solano et al., J. Sep Sci, 35(22):3124 (2012)). Proteins are expected to have minimal solubility at pH near the pi (Green, A.A., J. Biol. Chem., 93:517-542 (1931)). For SIESD.N103S (AFD.v14), increased solubility in PBS (pH 7.3) was not correlated with changes in pi. More precisely, the Asp to Ser amino acid changes in LC-CDR-L1 (VL-D28S and D31S) appear to alter the charge distribution on the surface of the molecule.
b.异构化和脱酰胺b. Isomerization and deamidation
为了模拟变体对在长效递送系统中可能发现的多种条件的暴露，在37℃在变化的pH和盐条件下对抗体进行应激达数周。具体地，在以下五种不同的制剂中对抗体进行评估：To mimic the exposure of the variants to the various conditions that might be found in long-acting delivery systems, antibodies were stressed at 37°C under varying pH and salt conditions for several weeks. Specifically, antibodies were evaluated in the following five different formulations:
制剂1：10mg/mL，10mM磷酸缓冲液，pH 2.5，Formulation 1: 10mg/mL, 10mM phosphate buffer, pH 2.5,
制剂2：10mg/mL，10mM组氨酸HCl，pH 5.5，Formulation 2: 10 mg/mL, 10 mM Histidine HCl, pH 5.5,
制剂3：10mg/mL，10mM磷酸缓冲液，pH 7.4(“低盐”)，Formulation 3: 10 mg/mL, 10 mM phosphate buffer, pH 7.4 ("low salt"),
制剂4：10mg/mL，pH 7.4PBS(“高盐”；10mM磷酸，137mM NaCl)；和Formulation 4: 10 mg/mL, pH 7.4 PBS ("high salt"; 10 mM phosphoric acid, 137 mM NaCl); and
制剂5：100mg/mL，pH 7.4PBSFormulation 5: 100 mg/mL, pH 7.4 PBS
所有溶液都具有0.02％PS20，在37℃孵育并且每2周取样一次。评估低盐条件(pH2.5、5.5和7.4)液体制剂中化学分解的作用。将PBS用作人玻璃体的pH和离子强度的模拟物。此外，将10mM磷酸盐，pH 7.4与PBS条件相比将揭示对化学和物理稳定性的离子强度作用。在孵育期间有规律地对PBS样品进行缓冲液交换以模仿玻璃体的交换。在所有5种条件下评估野生型AFD(“WT”或“aFD.WT”)和aVEGF。在除4号以外的所有制剂中测试D30E(AFD.v2)和TM(AFD.v6)变体。在制剂2和5中测试SIESD(AFD.v8)和SIESD.N103S(AFD.v14)。All solutions were with 0.02% PS20, incubated at 37°C and sampled every 2 weeks. The effect of chemical breakdown in liquid formulations under low salt conditions (pH 2.5, 5.5 and 7.4) was evaluated. PBS was used as a simulant for the pH and ionic strength of the human vitreous. Furthermore, comparing 10 mM phosphate, pH 7.4 to PBS conditions will reveal ionic strength effects on chemical and physical stability. The PBS samples were regularly buffer exchanged during the incubation period to mimic the exchange of the vitreous. Wild-type AFD ("WT" or "aFD.WT") and aVEGF were assessed under all five conditions. D30E (AFD.v2) and TM (AFD.v6) variants were tested in all formulations except No. 4. SIESD (AFD.v8) and SIESD.N103S (AFD.v14) were tested in Formulations 2 and 5.
被量化的化学分解是脱酰胺和异构化的脱水步骤，脱酰胺被表征为形成酸性峰，而异构化的脱水步骤被表征为形成长时间存在的长效琥珀酰亚胺(Asu)中间体，其作为碱性峰被检测到。使用阳离子交换色谱法(Dionex ProPac SAX-10columns)(IEC)来量化脱酰胺的Asn物质和脱水的Asp物质在不同制剂中的抗体样品内的出现。The quantified chemical breakdowns were the dehydration steps of deamidation characterized by the formation of acidic peaks and isomerization characterized by the formation of long-lived long-lived succinimide (Asu) intermediates body, which was detected as a basic peak. Cation exchange chromatography (Dionex ProPac SAX-10 columns) (IEC) was used to quantify the appearance of deamidated Asn species and dehydrated Asp species within antibody samples in different formulations.
对于所有测试的条件，在受试抗体中，aFD.WT显示主峰损失和碱性峰增加的最大速率。这在pH 5.5最明显，aVEGF、D30E(AFD.v2)、TM(AFD.v6)、SIESD(AFD.v8)和SIESD.N103S(AFD.v14)都显示比aFD.WT明显低的主峰损失速率，参见图3A。SIESD.N103S(AFD.v14)显示最慢的主峰损失速率，所述速率在pH 5.5与aVEGF相似(图3A)并且在PBS中甚至比aVEGF更慢(图3B)。如图4B中所示(100mg/ml的Fab，在PBS(pH 7.3)中)，D30E(AFD.v2)显示的碱性峰形成的速率为aFD.WT的约一半，而TM(AFD.v6)、SIESD(AFD.v8)和SIESD.N103S(AFD.14)显示可忽略的碱性峰形成。相反，如图4B中所示(100mg/ml的Fab，在PBS(pH 7.3)中)，对于PBS条件，aFD.WT和D30E(AFD.v2)的酸性峰产生的速率是相当的并且是对于TM(AFD.v6)和SIESD(AFD.v8)所确定的约2倍慢。PBS中SIESD.N103S(AFD.v14)的酸性峰形成基本上可忽略。For all conditions tested, aFD.WT showed the greatest rate of loss of the main peak and increase of the basic peak among the antibodies tested. This was most pronounced at pH 5.5, where aVEGF, D30E(AFD.v2), TM(AFD.v6), SIESD(AFD.v8) and SIESD.N103S(AFD.v14) all showed significantly lower loss rates of the main peak than aFD.WT , see Figure 3A. SIESD.N103S (AFD.v14) showed the slowest loss rate of the main peak, which was similar to aVEGF at pH 5.5 (Fig. 3A) and even slower than aVEGF in PBS (Fig. 3B). As shown in Figure 4B (Fab at 100 mg/ml in PBS (pH 7.3)), D30E (AFD.v2) displayed a rate of basic peak formation that was about half that of aFD.WT, while TM (AFD.v6 ), SIESD (AFD.v8) and SIESD.N103S (AFD.14) showed negligible basic peak formation. In contrast, as shown in Figure 4B (100 mg/ml of Fab in PBS (pH 7.3)), the rates of acidic peak generation for aFD.WT and D30E (AFD.v2) were comparable for PBS conditions and were the same as for About 2 times slower as determined by TM (AFD.v6) and SIESD (AFD.v8). The acidic peak formation of SIESD.N103S (AFD.v14) in PBS was essentially negligible.
c.聚集c. gather
利用尺寸排阻色谱法(SEC)来量化受试抗体的聚集体和单体的形成。使用的柱子是TSK-GEL Super SW2000(Tosoh Biosci.)。基于生产商的说明来使用材料和条件(www.tskgel.com)。Size exclusion chromatography (SEC) was used to quantify the formation of aggregates and monomers of the tested antibodies. The column used was TSK-GEL Super SW2000 (Tosoh Biosci.). Materials and conditions were used based on the manufacturer's instructions (www.tskgel.com).
在PBS中配制的100mg/ml的Fab的受试抗体的基于SEC数据的在37℃随时间的单体％显示在图5中。aFD.WT显示单体峰级分每月下降3-4％。与aFD.WT相比负电荷无变化的AFD.v2(D30E)显示相似的单体损失速率。AFD.v6、AFD.v8和AFD.v14显示下降的单体损失速率。第零天时单体含量的差异反映纯化的制剂在均一性方面的微小变化。在pH 5.5和7.4(无盐)在10mg/ml蛋白浓度下，D30E(AFD.v2)和TM(AFD.v6)的聚集速率是可比的。在pH 7.4添加盐不影响AFD.Ab变体的聚集速率但是其使aVEGF的聚集速率加倍。聚集依赖于蛋白浓度，因为在PBS中将浓度从10mg/mL增加至100mg/mL使得所有受试样品的聚集速率增加(表7)。在10mg/mL浓度在10mM磷酸缓冲液pH 7.4且无NaCl中，以及在10mg/mL浓度在PBS中的聚集是最小的(表7)。在37℃，在100mg/mL浓度在PBS中的aFD.WT和D30E(AFD.v2)的单体损失(在40天内分别为5.8％和7.3％)远大于在100mg/mL在PBS中的aVEGF、TM(AFD.v6)、SIESD(AFD.v8)和SIESD.N103S(AFD.v14)的单体损失(40天内分别为1.8％、1.5％、0.7％和1.5％)。这些数据暗示AFD.v6、AFD.v8和AFD.v14具有比aFD.WT和AFD.v2更少的聚集并且可以更适合作为治疗剂，因为其可以较少地倾向于体内免疫原性。The % monomer over time at 37° C. based on SEC data for 100 mg/ml of Fab formulated in PBS for the tested antibodies is shown in FIG. 5 . aFD.WT showed a 3-4% monthly decrease in the monomer peak fraction. AFD.v2(D30E) with no change in negative charge compared to aFD.WT showed a similar rate of monomer loss. AFD.v6, AFD.v8 and AFD.v14 showed decreased rates of monomer loss. Differences in monomer content at day zero reflect minor changes in the homogeneity of the purified preparations. The aggregation rates of D30E (AFD.v2) and TM (AFD.v6) were comparable at pH 5.5 and 7.4 (no salt) at a protein concentration of 10 mg/ml. Addition of salt at pH 7.4 did not affect the aggregation rate of the AFD.Ab variants but it doubled the aggregation rate of aVEGF. Aggregation was protein concentration dependent, as increasing the concentration from 10 mg/mL to 100 mg/mL in PBS increased the rate of aggregation for all samples tested (Table 7). Aggregation was minimal at 10 mg/mL concentration in 10 mM phosphate buffer pH 7.4 without NaCl, and at 10 mg/mL concentration in PBS (Table 7). At 37°C, the monomer losses of aFD.WT and D30E (AFD.v2) at a concentration of 100 mg/mL in PBS (5.8% and 7.3%, respectively, in 40 days) were much greater than that of aVEGF at 100 mg/mL in PBS , TM (AFD.v6), SIESD (AFD.v8) and SIESD.N103S (AFD.v14) monomer losses (1.8%, 1.5%, 0.7% and 1.5% over 40 days, respectively). These data suggest that AFD.v6, AFD.v8 and AFD.v14 have less aggregation than aFD.WT and AFD.v2 and may be more suitable as therapeutics as they may be less prone to immunogenicity in vivo.
表7.在40天通过SEC确定的盐和蛋白浓度对AFD.Ab变体和aVEGF的聚集的作用Table 7. Effect of salt and protein concentration on aggregation of AFD.Ab variants and aVEGF determined by SEC at 40 days
为检测作为pH的函数形成的碎片，进行毛细管电泳十二烷基硫酸钠(CE-SDS)，使用Beckman PA800System，利用无涂层的内径为50μm的熔融二氧化硅毛细管(PolymicroTechnologies,Inc)。通过Beckman Coulter NXp Liquid Handling Robot利用相当于Q12695的自动化来制备样品。以5kV的电压用15秒将样品注射到毛细管中，然后以15kV的电压固定30分钟。所有样品都在环境温度下运行。所有受试抗体的电泳图都与aFD.WT的电泳图类似。仅在pH 2.5观察到明显的碎片。在任何条件下都没有观察到更高分子量的物质，指示形成的任何聚集体都是可用SDS分解的并且不是共价相连的。To detect fragmentation as a function of pH, capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) was performed using a Beckman PA800 System utilizing uncoated 50 μm internal diameter fused silica capillaries (PolymicroTechnologies, Inc). Samples were prepared by a Beckman Coulter NXp Liquid Handling Robot with automation equivalent to Q12695. Samples were injected into the capillary at 5 kV for 15 seconds and then fixed at 15 kV for 30 minutes. All samples were run at ambient temperature. The electropherograms of all tested antibodies were similar to those of aFD.WT. Significant fragmentation was only observed at pH 2.5. No higher molecular weight species was observed under any condition, indicating that any aggregates formed were decomposable with SDS and were not covalently linked.
以上稳定性结果显示：抗-因子D的三重(TM(AFD.v6))和四重(SIESD(AFD.v8))突变体变体的化学稳定性与aFD.WT或D30E(AFD.v2)相比明显提高。在该系列中，SIESD.N103S(AFD.14)在pH 5.5和在PBS中具有最高的化学稳定性，类似于aVEGF的稳定性。异构化和脱酰胺位点都被去除并且在中性pH的溶解性提高，同时保持了fD结合亲和力。基于以上发现，本文中描述的所选的抗-因子D变体，尤其是SIESD(AFD.v8)和SIESD.N103S(AFD.v14)变体，适用于高浓度制剂和经由例如端口递送系统(PDS)设备的长效递送两者。例如，在生理条件的pH(～7.3)和离子强度(～150mM NaCl)下，利用耐久性的、可再填充的设备如端口递送系统的长效递送可能需要高浓度制剂和低的聚集倾向。The above stability results show that the chemical stability of anti-factor D triple (TM(AFD.v6)) and quadruple (SIESD(AFD.v8)) mutant variants is comparable to that of aFD.WT or D30E(AFD.v2) Significantly improved compared to. In this series, SIESD.N103S (AFD.14) had the highest chemical stability at pH 5.5 and in PBS, similar to the stability of aVEGF. Both isomerization and deamidation sites are removed and solubility at neutral pH is improved while maintaining fD binding affinity. Based on the above findings, selected anti-Factor D variants described herein, especially the SIESD (AFD.v8) and SIESD.N103S (AFD.v14) variants, are suitable for use in high concentration formulations and via, for example, port delivery systems ( PDS) long-acting delivery of both devices. For example, at physiological conditions of pH (-7.3) and ionic strength (-150 mM NaCl), long-term delivery using durable, refillable devices such as port delivery systems may require high concentration formulations and low aggregation propensity.
HVR序列列表(变体中的置换是加下划线的)List of HVR sequences (substitutions in variants are underlined)
实施例4：SIESD(AFD.v8)的高浓度制剂的稳定性Example 4: Stability of High Concentration Formulations of SIESD (AFD.v8)
基于以上稳定性研究，通过进行热(37℃)应力测试来评价AFD.v8对于低离子强度、pH 5.5缓冲液中的高浓度制剂的适用性。制备约100mg/mL AFD.v8的溶液，并且相对于pH 5.5的20mM组氨酸盐酸盐(His-HCl)透析，然后使用Amicon YM-10离心过滤器单元浓缩。使用280nm处的吸光度测量确定从过滤器单元移出后的蛋白质浓度为272mg/mL。使用灭菌的Spin-X(Costar)离心管过滤器通过0.22μm乙酸纤维素滤膜过滤100μL的等分试样。将包含在具有咬封盖的eppendorf管中的过滤后的溶液用石蜡膜密封并置于保持于37℃的温室中。在0、1、2、4和8个月的预定时间，取出试管，通过加入900μL储存缓冲液(10mM His-HCl，pH 5.5，10％海藻糖，0.01％聚山梨醇酯20)稀释10倍，并且冻存于-70℃直至可以进行分析。对于样品，通过离子交换色谱法(IEC)分析电荷变体的产生，通过尺寸排阻色谱法(SEC)分析聚集体的存在，通过表面等离子共振(SPR)测量分析抗原结合能力的保留，并且通过肽作图分析可变结构域中的具体化学变化。如实施例3(第00292段)中所述进行IEC，如实施例3(第00294段)中所述进行SEC。如下所述进行SPR和肽作图。Based on the stability studies above, the suitability of AFD.v8 for high concentration formulations in low ionic strength, pH 5.5 buffer was evaluated by performing a thermal (37°C) stress test. A solution of approximately 100 mg/mL AFD.v8 was prepared and dialyzed against 20 mM histidine hydrochloride (His-HCl) pH 5.5, then concentrated using an Amicon YM-10 centrifugal filter unit. The protein concentration after removal from the filter unit was determined to be 272 mg/mL using absorbance measurements at 280 nm. A 100 μL aliquot was filtered through a 0.22 μm cellulose acetate filter using a sterile Spin-X (Costar) centrifuge tube filter. The filtered solution contained in an eppendorf tube with a snap cap was sealed with parafilm and placed in a greenhouse maintained at 37°C. At predetermined times at 0, 1, 2, 4, and 8 months, remove the tubes and dilute 10-fold by adding 900 μL of storage buffer (10 mM His-HCl, pH 5.5, 10% trehalose, 0.01% polysorbate 20) , and stored frozen at -70°C until ready for analysis. For samples, the generation of charge variants was analyzed by ion exchange chromatography (IEC), the presence of aggregates by size exclusion chromatography (SEC), the retention of antigen-binding capacity by surface plasmon resonance (SPR) measurements, and by Peptide mapping analyzes specific chemical changes in the variable domains. IEC was performed as described in Example 3 (paragraph 00292) and SEC was performed as described in Example 3 (paragraph 00294). SPR and peptide mapping were performed as described below.
a.肽作图a. Peptide mapping
在1.5mL eppendorf管中用RCM缓冲液(6M盐酸胍，360mM Tris，2mM EDTA，pH8.6)将AFD.v8样品稀释至1mg/mL。通过加入1M二硫苏糖醇(DTT)至20mM的最终浓度，然后在37℃孵育1小时来引发还原反应。还原后，通过加入1M碘乙酸(IAA)至50mM的最终浓度并在室温下避光孵育15分钟来进行烷基化反应。使用G-25Minitrap柱，将还原和烷基化的样品缓冲液交换到消化缓冲液(25mM Tris,2.0M CaCl2,pH 8.2)中。以1:40的胰蛋白酶与蛋白质的比率(按质量计)加入胰蛋白酶，通过在37℃温育4小时使其消化完全。AFD.v8 samples were diluted to 1 mg/mL with RCM buffer (6M Guanidine HCl, 360 mM Tris, 2 mM EDTA, pH 8.6) in a 1.5 mL eppendorf tube. The reduction reaction was initiated by adding 1 M dithiothreitol (DTT) to a final concentration of 20 mM, followed by incubation at 37°C for 1 hour. After reduction, the alkylation reaction was carried out by adding 1 M iodoacetic acid (IAA) to a final concentration of 50 mM and incubating at room temperature in the dark for 15 minutes. The reduced and alkylated samples were buffer exchanged into digestion buffer (25 mM Tris, 2.0 M CaCl2 , pH 8.2) using a G-25 Minitrap column. Trypsin was added at a ratio of trypsin to protein (by mass) of 1:40, and the digestion was completed by incubation at 37°C for 4 hours.
LC/MS-MSLC/MS-MS
连接到Thermo Orbitrap Q Exactive质谱仪的Waters H-Class Acquity用LC/MS-MS分析。将10μg的胰蛋白酶消化的样品加载到Waters Acquity UPLC CSH柱上并使用以下LC条件运行：Analysis was by LC/MS-MS with a Waters H-Class Acquity connected to a Thermo Orbitrap Q Exactive mass spectrometer. Load 10 μg of the trypsinized sample onto a Waters Acquity UPLC CSH column and run using the following LC conditions:
流动相A–H20中的0.1％FAMobile Phase A – 0.1% FA inH2O
流动相B–ACN中的0.1％FAMobile phase B – 0.1% FA in ACN
柱温：77CColumn temperature: 77C
流动速率：0.2mL/minFlow rate: 0.2mL/min
对于质谱分析，使用FTMS(傅里叶变换MS，Orbitrap)以35K分辨率检测完整的MS1扫描。在全部MS1扫描中检测到的前8个离子(动态排除关闭)被选择用于在使用FTMS检测的数据依赖性MS2扫描中的HCD片段化。使用Thermo Scientific的XCalibur软件进行下游数据分析，包括提取离子色谱和定量天然和翻译后修饰的肽。For mass spectrometry, complete MS1 scans were detected at 35K resolution using FTMS (Fourier Transform MS, Orbitrap). The top 8 ions detected in all MS1 scans (dynamic exclusion turned off) were selected for HCD fragmentation in data-dependent MS2 scans using FTMS detection. Downstream data analysis, including extracted ion chromatography and quantification of native and post-translationally modified peptides, was performed using Thermo Scientific's XCalibur software.
b.通过SPR测量结合能力b. Measurement of binding capacity by SPR
通过SPR测量来评价结合固定的人因子D的功能活性。将S系列CM5传感器芯片对接到T200仪器1(GE Healthcare)中，用1X运行缓冲液引发，并且遵循制造商提供的方案用70％甘油进行标准化。传感器芯片表面使用具有制造商提供的材料和建议的方案的胺偶联试剂盒激活以用于抗原的胺偶联。在传感器芯片的流动池上，人因子D(fD)通过注射用10mM乙酸钠(pH 5)稀释fD(2.4mg/mL)制备的含有100μg/mL fD的溶液进行共价固定。流速为10μL/分钟，并且使用70μL的注射体积。这在fD的约5000共振单位(RU)的多次实验中产生了典型的偶联密度。通过注射70μL 1M乙醇胺封闭未反应的胺偶联位点。Functional activity of binding immobilized human Factor D was assessed by SPR measurement. Connect the S series CM5 sensor chip to In a T200 Instrument1 (GE Healthcare), prime with IX running buffer and normalize with 70% glycerol following the manufacturer's protocol. The sensor chip surface was activated for amine coupling of the antigen using an amine conjugation kit with materials and suggested protocols provided by the manufacturer. On the flow cell of the sensor chip, human factor D (fD) was covalently immobilized by injecting a solution containing 100 μg/mL fD prepared by diluting fD (2.4 mg/mL) with 10 mM sodium acetate (pH 5). The flow rate was 10 μL/min and an injection volume of 70 μL was used. This yielded typical coupling densities in multiple experiments at fD of about 5000 resonance units (RU). Block unreacted amine coupling sites by injecting 70 μL of 1 M ethanolamine.
使用T200评价软件的校准依赖性浓度分析程序确定AFD的抗原结合活性浓度。通过以下方法制备未受应力的AFD的标准曲线：将标准物重量稀释至5μg/mL，然后连续2倍稀释以产生2.5、1.25、0.625、0.313、0.156和0.078μg/mL的样品。通过重量稀释制备测试样品以获得约0.5、1.0或1.5μg/mL的蛋白质浓度。使用1X运行缓冲液(10mM HEPESpH 7.4，150mM NaCl，0.01％PS20)制备所有样品(200μL体积)。将60μL等分试样以10μL/分钟的流速注射到特异性抗原表面上，传感器芯片保持在25℃并且用1X运行缓冲液引发。在样品注射接近结束时由SPR信号确定与特异性抗原结合的抗体。在每个结合周期结束时通过注射30μL的10mM Gly-HCl pH 2.1来洗脱结合的抗体，以引起抗体-抗原复合物的解离。将起始材料的标准曲线用于确定SPR信号与抗体浓度之间的关系，使用四参数函数来分析数据。基于观察到的SPR信号，将由标准曲线计算的参数用于计算测试样品的抗原结合浓度。通过吸光度测量确定的该浓度与蛋白质浓度的比率给出了结合分数或百分比。use The calibration-dependent concentration analysis program of the T200 evaluation software determines the antigen-binding activity concentration of the AFD. A standard curve of unstressed AFD was prepared by diluting the standard weight to 5 μg/mL followed by serial 2-fold dilutions to generate samples at 2.5, 1.25, 0.625, 0.313, 0.156 and 0.078 μg/mL. Test samples were prepared by gravimetric dilution to achieve protein concentrations of approximately 0.5, 1.0 or 1.5 μg/mL. All samples (200 [mu]L volume) were prepared using IX running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.01% PS20). A 60 μL aliquot was injected onto the specific antigen surface at a flow rate of 10 μL/min, the sensor chip was kept at 25°C and primed with IX running buffer. Antibody binding to specific antigen was determined from the SPR signal near the end of sample injection. Bound antibody was eluted at the end of each binding cycle by injecting 30 μL of 10 mM Gly-HCl pH 2.1 to cause dissociation of the antibody-antigen complex. A standard curve of starting material was used to determine the relationship between SPR signal and antibody concentration, and a four-parameter function was used to analyze the data. Based on the observed SPR signal, the parameters calculated from the standard curve were used to calculate the antigen binding concentration of the test samples. The ratio of this concentration to the protein concentration, determined by absorbance measurement, gives the fraction or percentage bound.
c.结果c. Results
该热应力测试的结果表明AFD.v8是高浓度制剂中的稳定分子。在37℃下4个月后还保持了大于80％的抗原结合能力(图10A)。在4个月时发生非常少的聚集体形成，并且蛋白质为接近100％单体(通过SEC)(图10B)。酸性物质(图10B，％酸性-IEC)增加至约15％并且碱性物质增加至约20％(图10B，％碱性-IEC)表示4个月时发生的一些化学变化。在37℃下8个月后，酸性和碱性物质又有所增加，单体含量降低，并且因子D结合能力降低。肽作图表明酸性物质主要来源于CDR-H3Asn-103(根据Kabat编号的Asn-101)的脱酰胺作用(图10B，CDR-H3N101％脱酰胺作用)，而碱性变体是由N端处的焦谷氨酸形成(图10B，HC-E1％琥珀酰亚胺)和CDR-H3残基Glu-99(根据Kabat编号的Glu-95)(图10B，CDR-H3E97％异构化)的异构化所促成的。由于Asn-103和Glu-99(根据Kabat编号，分别为Asn-101和Glu-95)与lamaplizumab和因子D的共晶体结构中的因子D接触(Katschke KJ,Jr.,Wu P,Ganesan R,Kelley RF,Mathieu MA,Hass PE,Murray J,Kirchhofer D,Weismann C,van LookerenCampange M,“Inhibiting alternative pathway complement activation by targetingthe factor D exosite”,J.Biol.Chem.(2012)287:12886-92)，而重链的N-末端并非如此，所以Asn-103脱酰胺和Glu-99异构化以及单体含量降低可能直接导致在8个月时因子D结合的损失。尽管如此，由于这些化学和物理变化的速率缓慢，再加上预期在较低温度下速率进一步降低，储存于2-8℃或冷冻于-20℃的AFD.v8的高浓度液体制剂似乎将给出可接受的货架期。The results of this heat stress test indicate that AFD.v8 is a stable molecule in high concentration formulations. More than 80% of the antigen binding capacity was also retained after 4 months at 37°C (FIG. 10A). Very little aggregate formation occurred at 4 months, and the protein was nearly 100% monomeric (by SEC) (Fig. 10B). The increase in acidic species (Fig. 10B, % Acidic-IEC) to about 15% and alkaline species to about 20% (Fig. 10B, % Basic-IEC) indicated some chemical changes that occurred at 4 months. After 8 months at 37°C, the acidic and basic substances increased again, the monomer content decreased, and the factor D binding ability decreased. Peptide mapping indicated that the acidic species was mainly derived from deamidation of CDR-H3Asn-103 (Asn-101 according to Kabat numbering) (Figure 10B, CDR-H3N101% deamidation), while the basic variant was derived from Pyroglutamate formation (Fig. 10B, HC-E 1% succinimide) and CDR-H3 residue Glu-99 (Glu-95 according to Kabat numbering) (Fig. 10B, CDR-H3E 97% isomerization) caused by isomerization. Since Asn-103 and Glu-99 (Asn-101 and Glu-95, respectively, according to Kabat numbering) are in contact with Factor D in the co-crystal structure of lamaplizumab and Factor D (Katschke KJ, Jr., Wu P, Ganesan R, Kelley RF, Mathieu MA, Hass PE, Murray J, Kirchhofer D, Weismann C, van LookerenCampange M, “Inhibiting alternative pathway complement activation by targeting the factor D exosite”, J.Biol.Chem.(2012)287:12886-92) , while this was not the case for the N-terminus of the heavy chain, so deamidation of Asn-103 and isomerization of Glu-99 and reduced monomer content may directly contribute to the loss of Factor D binding at 8 months. Nevertheless, due to the slow rate of these chemical and physical changes, coupled with the expected further decrease in rate at lower temperatures, it appears that highly concentrated liquid formulations of AFD.v8 stored at 2-8°C or frozen at -20°C will give acceptable shelf life.
实施例5：AFD.v8/v14的兔pKExample 5: Rabbit pK of AFD.v8/v14
在兔中进行AFD.v8和AFD.v14的体内pK研究。由单剂量实验确定pK参数，因为在重复用药后或在通过持续递送制剂增加暴露时，人源化抗体在兔中有免疫原性。In vivo pK studies of AFD.v8 and AFD.v14 were performed in rabbits. The pK parameters were determined from single-dose experiments because the humanized antibody was immunogenic in rabbits after repeated dosing or when exposure was increased by sustained delivery formulations.
动物的护理遵循Genentech Institutional动物护理和使用委员会指南。将天然新西兰白(NZW)兔(41只雄性动物；3.1kg至4.1kg并且在用药时为约4月龄)分配到各剂量组并且利用在Charles River Laboratories的测试项目进行用药。Animal care followed Genentech Institutional Animal Care and Use Committee guidelines. Native New Zealand White (NZW) rabbits (41 male animals; 3.1 kg to 4.1 kg and approximately 4 months of age at the time of dosing) were assigned to dose groups and dosed using the test program at Charles River Laboratories.
经由单次双侧玻璃体内注射将SIESD(AFD.v8)、SIESD.N103S(AFD.v14)或雷珠单抗施用至兔并且进行观察达27天。在治疗前一天，紧接着注射后，将局部抗生素(托普霉素眼膏)两次施用于双眼，并且在注射后一天施用两次，在第1天和第2天送去验尸的动物除外。在用药前，将散瞳剂液滴(1％托吡卡胺)施用于每只眼以进行充分扩瞳。在所述过程之前或期间用异氟醚/氧气使动物镇静。还在注射前将爱尔卡因(0.5％)施用于每只眼。用稀释在无菌水中的苯扎氯铵(ZephiranTM)冲洗结膜，U.S.P.至1:10,000(v/v)。SIESD (AFD.v8), SIESD.N103S (AFD.v14) or ranibizumab were administered to rabbits via a single bilateral intravitreal injection and observed for 27 days. A topical antibiotic (tobramycin ophthalmic ointment) was applied twice to both eyes the day before treatment, immediately after the injection, and twice the day after the injection, except for animals sent for necropsy on days 1 and 2 . Prior to dosing, mydriatic drops (1% tropicamide) were applied to each eye for adequate pupil dilation. Animals were sedated with isoflurane/oxygen before or during the procedure. Alcaine (0.5%) was also administered to each eye prior to injection. The conjunctiva was irrigated with benzalkonium chloride (ZephiranTM) diluted in sterile water, U.S.P. to 1:10,000 (v/v).
在快要用药前在层流柜中充填注射器。通过单次30μL玻璃体内注射(0.3mg剂量)将Fab施用于所有动物的双眼。由委员会认证的兽医眼科医生利用消毒的带有30规格x 1/2”针头的100μL Hamilton Luer Lock注射器来施用剂量。为了模拟临床用药，在下前方象限，即对于左眼和右眼，分别在5点钟和7点钟位置(当面向动物时)，给眼睛用药。在处理后立即通过裂隙灯活组织显微镜检查和/或间接的检眼镜检查对眼睛进行检查。Fill the syringes in the laminar flow cabinet shortly before dosing. Fab was administered to both eyes of all animals by a single 30 μL intravitreal injection (0.3 mg dose). Doses were administered by a board-certified veterinary ophthalmologist using sterile 100 μL Hamilton Luer Lock syringes with 30 gauge x 1/2" needles. To simulate clinical dosing, doses were administered in the lower anterior quadrant, ie, for the left and right eyes, at 5 Eyes were administered at the o'clock and 7 o'clock positions (when facing the animal.) Eyes were examined by slit lamp biomicroscopy and/or indirect ophthalmoscopy immediately after treatment.
在通过静脉内注射戊巴比妥钠进行麻醉后，通过切割腋动脉或股动脉对所有动物进行放血。收集房水、玻璃体液和视网膜组织，在液氮中速冻并且存储在-80℃。通过在50mMTris-HCl pH 8.0，1M NaCl中的匀浆化提取视网膜中的抗体Fab。通过以下描述的GRIPELISA确定测试品的玻璃体和视网膜浓度。低于LLOQ的值不被用于药物动力学分析或用于绘图或概述。通过非区室分析利用标称时间和剂量来确定药物动力学参数(PhoenixWinNonlin,Pharsight Corp,Mountain View,CA)。After anesthesia by intravenous injection of sodium pentobarbital, all animals were exsanguinated by cutting the axillary or femoral artery. Aqueous humor, vitreous humor and retinal tissue were collected, snap frozen in liquid nitrogen and stored at -80°C. Antibody Fabs in the retina were extracted by homogenization in 50 mM Tris-HCl pH 8.0, 1 M NaCl. The vitreous and retinal concentrations of the test articles were determined by GRIPELISA as described below. Values below the LLOQ were not used for pharmacokinetic analysis or for plotting or summarizing. Pharmacokinetic parameters were determined by noncompartmental analysis using nominal time and dose (Phoenix WinNonlin, Pharsight Corp, Mountain View, CA).
在通用免疫球蛋白药物动力学(GRIP)ELISA中进行对SIESD(AFD.v8)、SIESD.N103S(AFD.v14)和雷珠单抗的分析，本文所述除外。将绵羊抗-人-IgG(The BindingSite；San Diego，CA)稀释至1000ng/mL，在0.5M碳酸盐/碳酸氢盐，pH 9.6中，并且在4℃过夜孵育期间涂覆在384孔ELISA平板(Nunc；Neptune，NJ)上。将平板用PBS加上0.05％Tween-20洗涤并且在与PBS加上0.5％牛血清白蛋白(BSA)一起孵育1至2小时期间进行封闭。该温育和所有后续温育都在温和的搅拌下在室温进行。通过在测定缓冲液(PBS，0.5％BSA，15ppm Proclin，0.05％Tween 20，0.25％CHAPS，5mM EDTA，0.35M NaCl，(pH 7.4))中40-0.625ng/mL连续稀释AFD.v8、AFD.v14或雷珠单抗来制备标准曲线。兔玻璃体或视网膜匀浆样品分别在测定缓冲液中进行最小1:100或1:50稀释。然后将稀释的标准物、对照及样品在清洗过的平板上孵育1-2小时。在洗涤步骤后，在利用在测定稀释液(PBS+0.5％BSA+0.05％Tween 20+10ppm Proclin)中的稀释至83.3ng/mL的缀合了HRP的绵羊抗-人IgG mAb(Bethyl Laboratories Inc；Montgomery，TX)的1.5小时孵育期间检测与平板结合的AFD.v8、AFD.v14或雷珠单抗。在最后洗涤后，添加四甲基联苯胺过氧化物酶底物(Moss,Inc.,Pasadena,MD)，显色10-15分钟，并且用1M磷酸终止反应。使用酶标仪(MultiscanAscent,Thermo Fischer；Waltham,MA)在450nm对平板进行读数，以620nm作为参比。使用内部的基于Excel的软件，由相应的标准曲线的四参数拟合计算AFD.v8、AFD.v14或雷珠单抗的浓度。考虑到玻璃体或视网膜匀浆中的最小稀释，AFD.v8、AFD.v14或雷珠单抗在兔玻璃体或视网膜匀浆中的最小可量化浓度分别为62.5ng/mL或31.25ng/mL。Analysis of SIESD (AFD.v8), SIESD.N103S (AFD.v14) and ranibizumab was performed in a general immunoglobulin pharmacokinetic (GRIP) ELISA, except as described herein. Sheep anti-human-IgG (The BindingSite; San Diego, CA) was diluted to 1000 ng/mL in 0.5M carbonate/bicarbonate, pH 9.6, and coated in a 384-well ELISA during an overnight incubation at 4°C. on a plate (Nunc; Neptune, NJ). Plates were washed with PBS plus 0.05% Tween-20 and blocked during a 1-2 hour incubation with PBS plus 0.5% bovine serum albumin (BSA). This and all subsequent incubations were performed at room temperature with gentle agitation. AFD.v8, AFD was serially diluted at 40-0.625 ng/mL in assay buffer (PBS, 0.5% BSA, 15ppm Proclin, 0.05% Tween 20, 0.25% CHAPS, 5mM EDTA, 0.35M NaCl, (pH 7.4)) .v14 or ranibizumab to prepare the standard curve. Rabbit vitreous or retinal homogenate samples were diluted in assay buffer at a minimum of 1:100 or 1:50, respectively. Diluted standards, controls and samples were then incubated on the washed plates for 1-2 hours. After the washing step, HRP-conjugated sheep anti-human IgG mAb (Bethyl Laboratories Inc AFD.v8, AFD.v14, or ranibizumab bound to the plate was detected during the 1.5-hour incubation period in Montgomery, TX). After the final wash, tetramethylbenzidine peroxidase substrate (Moss, Inc., Pasadena, MD) was added, the color developed for 10-15 minutes, and the reaction was stopped with 1M phosphoric acid. Plates were read using a microplate reader (Multiscan Ascent, Thermo Fischer; Waltham, MA) at 450 nm with a reference of 620 nm. Concentrations of AFD.v8, AFD.v14, or ranibizumab were calculated from four-parameter fits of the corresponding standard curves using in-house Excel-based software. Considering minimal dilution in vitreous or retinal homogenates, the minimum quantifiable concentrations of AFD.v8, AFD.v14, or ranibizumab in rabbit vitreous or retinal homogenates were 62.5 ng/mL or 31.25 ng/mL, respectively.
玻璃体内注射0.3mg SIESD(AFD.v8)、SIESD.N103S(AFD.v14)或比较剂量的雷珠单抗(抗-VEGF)观察到的时间依赖性浓度曲线显示在图11中。The time-dependent concentration profiles observed for intravitreal injections of 0.3 mg SIESD (AFD.v8), SIESD.N103S (AFD.v14) or comparative doses of ranibizumab (anti-VEGF) are shown in FIG. 11 .
使用非区室模型的玻璃体数据分析指示SIESD(AFD.v8)和SIESD.N103S(AFD.v14)两者都具有与雷珠单抗非常相似的清除性质。在三眼室：玻璃体液、房水和视网膜中，三种蛋白质全部给出非常相似的暴露，如AUC参数反映的。计算的雷珠单抗的PK参数与之前在兔子中的研究的结果一致(Gaudrealt等,Retina,27:1260-6(2007))。SIESD(AFD.v8)和SIESD.N103S(AFD.v14)都显示不依赖于靶标的眼清除性质，这使得这些分子适合开发。Analysis of vitreous data using a non-compartmental model indicated that both SIESD (AFD.v8) and SIESD.N103S (AFD.v14) had very similar clearance properties to ranibizumab. In the third eye compartment: vitreous humor, aqueous humor and retina, all three proteins gave very similar exposures, as reflected by the AUC parameters. The calculated PK parameters of ranibizumab were consistent with the results of previous studies in rabbits (Gaudrealt et al., Retina, 27:1260-6 (2007)). Both SIESD (AFD.v8) and SIESD.N103S (AFD.v14) display target-independent ocular clearance properties, which makes these molecules suitable for development.
实施例6：AFD.v8/v14的粘度Example 6: Viscosity of AFD.v8/v14
因为低粘度对于玻璃体内给药是重要的，因此在不同蛋白浓度在pH5.5，低盐缓冲液中测量SIESD(AFD.v8)和SIESD.N103S(AFD.v14)的粘度。使用1000s-1的剪切速率在恒温于25℃的TA Instruments锥板流变仪上进行粘度测量。Because low viscosity is important for intravitreal administration, the viscosity of SIESD (AFD.v8) and SIESD.N103S (AFD.v14) was measured at different protein concentrations in pH 5.5, low salt buffer. Viscosity measurements were performed on a TA Instruments cone and plate rheometer thermostatted at 25 °C using a shear rate of 1000 s−1 .
aFD.WT、SIESD(AFD.v8)和SIESD.N103S(AFD.v14)给出类似的依赖于蛋白浓度的粘度曲线，其中即使在超过200mg/mL的浓度，粘度对于玻璃体内注射来说也是可接受的(<30cP)(图12)。aFD.WT, SIESD (AFD.v8) and SIESD.N103S (AFD.v14) gave similar protein concentration dependent viscosity curves, where even at concentrations exceeding 200 mg/mL the viscosity was acceptable for intravitreal injection Acceptable (<30cP) (Figure 12).
实施例7.进一步修饰抗-因子D抗体变体用于聚合物缀合物Example 7. Further modification of anti-Factor D antibody variants for use in polymer conjugates
以上实施例中所述的aFD.WT和变体是Fab片段。虽然其轻链和重链的可变结构域(VL和VH)序列不同，如图1B中所示，但其恒定结构域CL和CH1保持相同。特别地，如图1A(SEQID NO:2)、图1C(SEQ ID NO:27)和图1D(SEQ ID NO:29)中所示，重链的CH1结构域终止于苏氨酸残基。为了制备用于聚合物缀合如PEG化的AFD.Ab变体，通过添加自Fab’对应物的铰链区起的第一个半胱氨酸残基将Fab片段的重链进一步修饰(例如，对于AFD.v8为Cys-修饰的HC(Fab-C)(SEQ ID NO:30)，对于AFD.v14为Cys-修饰的HC(Fab-C)(SEQ ID NO:32))，使得添加的半胱氨酸充当PEG聚合物的连接位点。所得的片段可以因此与多臂PEG的一个臂缀合。还通过添加自Fab’对应物的铰链区起的头四个残基，即Cys-Pro-Pro-Cys(SEQ ID NO:21)来修饰Fab片段的重链(例如对于AFD.v8为Cys-Pro-Pro-Cys-修饰的HC(SEQ ID NO:31)，对于AFD.v14为Cys-Pro-Pro-Cys-修饰的HC(SEQ ID NO:33))，使得添加的两个Cys都充当PEG的连接位点，导致修饰的AFD.Ab片段能够连接两个PEG分子。aFD.WT and variants described in the above examples are Fab fragments. Although the variable domain (VL and VH) sequences of its light and heavy chains are different, as shown in Figure 1B, its constant domains, CL and CH1, remain the same. In particular, the CH1 domain of the heavy chain terminates at a threonine residue as shown in Figure 1A (SEQ ID NO:2), Figure 1C (SEQ ID NO:27) and Figure ID (SEQ ID NO:29). To prepare AFD.Ab variants for polymer conjugation such as PEGylation, the heavy chain of the Fab fragment is further modified by adding the first cysteine residue from the hinge region of the Fab' counterpart (e.g., Cys-modified HC (Fab-C) (SEQ ID NO:30) for AFD.v8 and Cys-modified HC (Fab-C) (SEQ ID NO:32) for AFD.v14, such that the added Cysteine serves as the attachment site for the PEG polymer. The resulting fragment can thus be conjugated to one arm of a multi-armed PEG. The heavy chain of the Fab fragment was also modified by adding the first four residues from the hinge region of the Fab' counterpart, Cys-Pro-Pro-Cys (SEQ ID NO: 21) (eg Cys-Pro-Cys for AFD.v8). Pro-Pro-Cys-modified HC (SEQ ID NO:31), for AFD.v14 is Cys-Pro-Pro-Cys-modified HC (SEQ ID NO:33)), so that both Cys added act as A linking site for PEG, resulting in the ability of the modified AFD.Ab fragment to link two PEG molecules.
遵循随试剂盒提供的方案，使用(Agilent)诱变试剂盒制备Cys-修饰的和Cys-Pro-Pro-Cys-修饰的变体。合成载有所需的密码子改变的寡核苷酸引物。通过DNA测序鉴定和确定具有设计的改变的质粒。对于小规模表达，将DNA转化到大肠杆菌菌株64B4中。将单菌落挑取到含有50μg/mL羧苄青霉素(carbenecillin)(培养基制备代码A3232)的5mL LB培养基(培养基制备编码A2008)中，并且在14mL培养管中在以200RPM进行振荡的情况下在Innova培养箱中在37℃培养过夜。将这些培养物用于接种在1L带挡板的摇瓶中的250mL的完全豆粕(soy crap)培养基(培养基制备编码A4564)(50μg/mL羧苄青霉素)中。将培养物在30℃在以200RPM进行振荡的情况下培养过夜，然后通过离心收集。如实施例1所描述的，将细胞沉淀用PopCulture介质(Invitrogen)裂解，并且纯化Fab。对于较大规模的Fab-C制备，将来自10L转化细胞发酵的细胞糊悬浮在提取缓冲液中并且使用微型流化仪将其匀浆化，并且如实施例8所描述的对Fab-C进行纯化。Following the protocol provided with the kit, use (Agilent) mutagenesis kits to prepare Cys-modified and Cys-Pro-Pro-Cys-modified variants. Oligonucleotide primers carrying the desired codon changes were synthesized. Plasmids with designed changes were identified and confirmed by DNA sequencing. For small-scale expression, the DNA was transformed into E. coli strain 64B4. Pick a single colony into 5 mL of LB medium (medium preparation code A2008) containing 50 μg/mL carbenecillin (medium preparation code A3232) and shake at 200 RPM in a 14 mL culture tube Incubate overnight at 37°C in an Innova incubator. These cultures were used to inoculate 250 mL of complete soy crap medium (media preparation code A4564) (50 μg/mL carbenicillin) in 1 L baffled shake flasks. Cultures were grown overnight at 30°C with shaking at 200 RPM, then harvested by centrifugation. Cell pellets were lysed with PopCulture medium (Invitrogen) and Fab purified as described in Example 1 . For larger-scale Fab-C preparation, cell paste from 10 L of transformed cell fermentation was suspended in extraction buffer and homogenized using a microfluidizer, and Fab-C was processed as described in Example 8. purification.
实施例8：AFD.v14缀合物的制备Example 8: Preparation of AFD.v14 conjugates
将实施例7中制备的含有Cys修饰的HC(SEQ ID NO:32)的AFD.v14变体(本文中称为“Cys修饰的AFD.v14变体”或“AFD.v14.C”)与具有不同核心结构的市售马来酰亚胺官能化的多臂PEG缀合。The AFD.v14 variant containing Cys-modified HC (SEQ ID NO:32) prepared in Example 7 (referred to herein as "Cys-modified AFD.v14 variant" or "AFD.v14.C") was combined with Commercially available maleimide-functionalized multi-armed PEG conjugations with different core structures.
a.马来酰亚胺官能化的多臂PEGa. Maleimide-functionalized multi-arm PEG
将下表8中详述的马来酰亚胺官能化的多臂PEG用于缀合反应中：The maleimide-functionalized multi-armed PEGs detailed in Table 8 below were used in the conjugation reactions:
使用MALDI对8ARM(TP)-PEG-MAL(JenKem Technology,USA)和HGEO-400MA(NOF America,Corp.)进行分析，以比较含有TP核心或HGEO核心的PEG八聚体的均匀性。结果显示在图13A和13B中。8ARM(TP)-PEG-MAL (JenKem Technology, USA) and HGEO-400MA (NOF America, Corp.) was analyzed to compare the homogeneity of PEG octamers containing TP core or HGEO core. The results are shown in Figures 13A and 13B.
从图13A和13B可以看出，含有TP核心的8ARM(TP)-PEG-MAL比含有HGEO核心的HGEO-400MA更均匀。As can be seen from Figures 13A and 13B, the 8ARM(TP)-PEG-MAL containing the TP core was more HGEO-400MA is more uniform.
b.Cys修饰的AFD.v14变体与马来酰亚胺官能化的多臂PEG的缀合b. Conjugation of Cys-modified AFD.v14 variants to maleimide-functionalized multi-armed PEG
使用Gamma Plus树脂捕获实施例7中制备的Cys修饰的AFD.v14变体，用6.5mM GSHpH 8.5洗涤5个柱体积以将c-末端半胱氨酸去封闭并破坏Fab-C二聚体形成，接着洗脱到0.1M乙酸(pH 2.9)中。使用SP Sepharose高效强阳离子交换树脂(来自GE Healthcare)，在25mM乙酸钠(pH 5.0)中，用0.05％Triton X-100+0.05％Triton X-114将Cys修饰的AFD.v14单体进一步分离19小时以去除内毒素。在20个柱体积内，使用0-20％25mM乙酸钠(pH 5.0)+1M NaCl之间的梯度进行洗脱。然后通过使用1M HEPES pH 7.2滴定至pH 6.5来制备具有去封闭的c-末端半胱氨酸的单体Fab-C以用于PEG化。然后，将Cys修饰的AFD.v14Fab-C以约5mg/mL的浓度在25mM乙酸钠(pH 6.5)、150mM NaCl、4mM EDTA中与马来酰亚胺功能化的多臂PEG缀合。Cys修饰的AFD.v14变体未被进一步浓缩以使由于Fab-C二聚化引起的半胱氨酸反应性损失最小化。在平衡至室温后，将马来酰亚胺功能化的多臂PEG重悬于25mM乙酸钠(pH5.0)中至10mg/mL的浓度。将pH保持在低于pH 6以避免马来酰亚胺开环。一旦PEG溶解，将其以0.1125:1的PEG与Fab-C的摩尔比添加至Fab-C池中。然后将混合物在温和的震荡下在室温过夜。第二天，通过SEC-MALS检查缀合效率(#Fab/PEG)。Cys-modified AFD.v14 variants prepared in Example 7 were captured using Gamma Plus resin, washed with 6.5 mM GSH pH 8.5 for 5 column volumes to deblock the c-terminal cysteine and disrupt Fab-C dimer formation , followed by elution into 0.1 M acetic acid (pH 2.9). Cys-modified AFD.v14 monomers were further isolated with 0.05% Triton X-100 + 0.05% Triton X-114 using SP Sepharose high-efficiency strong cation exchange resin (from GE Healthcare) in 25 mM sodium acetate (pH 5.0)19 hours to remove endotoxin. Elution was performed using a gradient between 0-20% 25 mM sodium acetate (pH 5.0) + 1 M NaCl over 20 column volumes. Monomeric Fab-C with deblocked c-terminal cysteine was then prepared for PEGylation by titrating to pH 6.5 using 1 M HEPES pH 7.2. Cys-modified AFD.v14Fab-C was then conjugated to maleimide-functionalized multi-armed PEG at a concentration of approximately 5 mg/mL in 25 mM sodium acetate (pH 6.5), 150 mM NaCl, 4 mM EDTA. Cys-modified AFD.v14 variants were not further condensed to minimize loss of cysteine reactivity due to Fab-C dimerization. After equilibrating to room temperature, the maleimide-functionalized multi-armed PEG was resuspended in 25 mM sodium acetate (pH 5.0) to a concentration of 10 mg/mL. The pH was kept below pH 6 to avoid ring opening of the maleimide. Once the PEG was dissolved, it was added to the Fab-C pool at a molar ratio of PEG to Fab-C of 0.1125:1. The mixture was then left overnight at room temperature with gentle shaking. The next day, the conjugation efficiency (#Fab/PEG) was checked by SEC-MALS.
实施例9：AFD.v14缀合物的纯化和表征Example 9: Purification and Characterization of AFD.v14 Conjugates
纯化实施例8中制备的缀合物并使用SEC-MALS分析以确认PEG化并确定不同PEG核心结构的缀合效率。除非另有指明，使用在等度条件下使用磷酸盐缓冲盐水(PBS)(pH7.2)、150mM NaCl以0.8mL/分钟运行的300x 8mm Shodex OH pak SB-804HQ，通过尺寸排阻色谱(SEC)确定缀合效率。摩尔质量使用Wyatt Technology的直列静态多角度激光散射(MALS)确定。将光子相关光谱用于确定流体动力学半径(RH)，使用准弹性光散射(QELS)，即具有99.0°处检测的单光子计数模块(也属于Wyatt Technology)。使用Wyatt所有的Astra软件处理原始数据，其中使用利妥昔单抗标准品设定摩尔质量和RH常数。The conjugate prepared in Example 8 was purified and analyzed using SEC-MALS to confirm PEGylation and determine the conjugation efficiency of different PEG core structures. Unless otherwise indicated, the samples were analyzed by size exclusion chromatography (SEC) using a 300x 8 mm Shodex OH pak SB-804HQ run at 0.8 mL/min under isocratic conditions using phosphate-buffered saline (PBS) (pH 7.2), 150 mM NaCl ) to determine conjugation efficiency. Molar masses were determined using Wyatt Technology's Inline Static Multi-Angle Laser Scattering (MALS). Photon correlation spectroscopy was used to determine the hydrodynamic radius (RH) using quasi-elastic light scattering (QELS), a single photon counting module (also of Wyatt Technology) with detection at 99.0°. Raw data were processed using Astra software owned by Wyatt, where molar masses and RH constants were set using rituximab standards.
a.Cys修饰的AFD.v14-8ARM(TP)-PEG-MAL缀合物a.Cys-modified AFD.v14-8ARM(TP)-PEG-MAL conjugate
在20mM His-乙酸盐(pH 5.5)、50mM NaCl(等度梯度)中，在Sephacryl S-300HR(GE Healthcare)柱上使用SEC纯化实施例8中制备的Cys修饰的AFD.v14-8ARM(TP)-PEG-MAL缀合物(含有TP核心结构)(下文中称为“AFD.v14TP缀合物”或“AFD.v14.C+TP八聚体”)。使用Wyatt Technology的直列静态多角度激光散射(MALS)和Shodex OH pak SB-804，确定摩尔质量和缀合效率(图14C)。使用Wyatt所有的Astra软件分析原始数据，其中使用利妥昔单抗标准品设定摩尔质量常数。摩尔质量用于估计与每个PEG连接的AFD.v14变体的平均数量。结果显示于图14A、14B和14C以及表9中。The Cys-modified AFD.v14-8ARM prepared in Example 8 was purified using SEC on a Sephacryl S-300HR (GE Healthcare) column in 20 mM His-acetate (pH 5.5), 50 mM NaCl (isocratic gradient) ( TP)-PEG-MAL conjugate (containing TP core structure) (hereinafter referred to as "AFD.v14TP conjugate" or "AFD.v14.C+TP octamer"). Molar masses and conjugation efficiencies were determined using Wyatt Technology's in-line static multi-angle laser scattering (MALS) and Shodex OH pak SB-804 (Figure 14C). Raw data were analyzed using Astra software owned by Wyatt, where molar mass constants were set using rituximab standards. Molar mass was used to estimate the average number of AFD.v14 variants attached to each PEG. The results are shown in Figures 14A, 14B and 14C and Table 9.
表9Table 9
agg＝聚集物；n/d＝未确定agg = aggregate; n/d = not determined
*B5-B7视为MALS测量中基于误差％的8个Fab/PEG。*B5-B7 are considered as 8 Fab/PEG based on error % in MALS measurement.
从表9中可以看出，Cys修饰的AFD.v14变体(AFD.v14.C)与具有TP核心的多臂PEG八聚体的缀合产生了包含8个Fab/PEG的缀合物，这表明使用包含TP核心的PEG八聚体可以实现良好的缀合效率(例如，8个Fab/PEG从缀合的约45％回收)。As can be seen in Table 9, conjugation of a Cys-modified AFD.v14 variant (AFD.v14.C) to a multi-armed PEG octamer with a TP core yielded a conjugate comprising eight Fab/PEGs, This suggests that good conjugation efficiencies can be achieved using PEG octamers comprising a TP core (eg, 8 Fab/PEG recovered from about 45% of conjugation).
b.Cys修饰的AFD.v14-8ARM-PEG-MAL缀合物b.Cys modified AFD.v14-8ARM-PEG-MAL conjugate
在20mM His-乙酸盐(pH 5.5)、50mM NaCl(等度梯度)中，在Sephacryl S-300HR(GE Healthcare)柱上使用SEC纯化实施例8中制备的Cys修饰的AFD.v14-8ARM-PEG-MAL缀合物(含有HG核心结构(JenKem))(下文中称为“AFD.v14HG缀合物”或“AFD.v14.C+HG八聚体”)。使用Tosoh G3000PW柱和Wyatt Technology的直列静电MALS确定摩尔质量和缀合效率。将光子相关光谱用于确定流体动力学半径(RH)，使用准弹性光散射(QELS)，即具有99°处检测的单光子计数模块(也属于Wyatt Technology)。使用Wyatt所有的Astra软件分析原始数据，其中使用利妥昔单抗标准品设定摩尔质量和RH常数。摩尔质量用于估计与每个PEG连接的AFD.v14变体的数量。结果显示于图15A、15B和15C以及表10中。The Cys-modified AFD.v14-8ARM- PEG-MAL conjugate (containing HG core structure (JenKem)) (hereinafter referred to as "AFD.v14HG conjugate" or "AFD.v14.C+HG octamer"). Molar masses and conjugation efficiencies were determined using Tosoh G3000PW columns and inline electrostatic MALS from Wyatt Technology. Photon correlation spectroscopy was used to determine the hydrodynamic radius (RH) using quasi-elastic light scattering (QELS), a single photon counting module with detection at 99° (also belonging to Wyatt Technology). Raw data were analyzed using Astra software owned by Wyatt, with rituximab standards used to set molar masses and RH constants. Molar mass was used to estimate the number of AFD.v14 variants attached to each PEG. The results are shown in Figures 15A, 15B and 15C and Table 10.
表10Table 10
agg＝聚集物；n/d＝未确定agg = aggregate; n/d = not determined
从表10中可以看出，Cys修饰的AFD.v14变体(AFD.v14.C)与包含HG核心的PEG八聚体的缀合产生了包含8个Fab/PEG的缀合物。然而，包含8个Fab/PEG的缀合物的回收率(约20％回收)约为使用具有TP核心的缀合物时包含8个Fab/PEG的缀合物的量的一半。相较于与TP核心的缀合所观察到的，与HG核心的缀合还产生了更多的包含5-7个Fab/PEG的缀合物，以及显著更多的聚集体。As can be seen in Table 10, conjugation of a Cys-modified AFD.v14 variant (AFD.v14.C) to a PEG octamer comprising a HG core resulted in a conjugate comprising 8 Fab/PEGs. However, the recovery of the conjugate comprising 8 Fab/PEG (approximately 20% recovery) was about half of the amount of conjugate comprising 8 Fab/PEG when using the conjugate with TP core. Conjugation to the HG core also produced more conjugates containing 5-7 Fab/PEG, and significantly more aggregates than observed with the TP core.
为了改善Fab化的估计和RH测量，将含有S-300纯化后获得的级分的产物汇集并备选地在PBS(pH 7.4)中在10/300Sephacryl S-400HR(GE Healthcare)柱上使用SEC-MALS进行分析，其以0.25mL/分钟运行。如上所描述的确定摩尔质量和RH。SEC和MALS结果显示在图16A和16B中。To improve estimation of Fab and RH measurement, products containing fractions obtained after S-300 purification were pooled and alternatively used SEC on a 10/300 Sephacryl S-400HR (GE Healthcare) column in PBS (pH 7.4) -MALS for analysis, which runs at 0.25 mL/min. Molar masses and RH were determined as described above. SEC and MALS results are shown in Figures 16A and 16B.
使用8ARM-PEG-MAL(HG核心)制备并使用Sephacryl S-400HR分析的缀合物具有12.2nm(±4.5％)的平均RH，340.3kDa(±8.9％)的平均摩尔质量，和6.4个Fab/PEG的平均值。The conjugate prepared using 8ARM-PEG-MAL (HG core) and analyzed using Sephacryl S-400HR had an average RH of 12.2 nm (±4.5%), an average molar mass of 340.3 kDa (±8.9%), and 6.4 Fab /PEG average.
b.Cys修饰的AFD.v14-HGEO-400MA缀合物b.Cys modified AFD.v14-HGEO-400MA conjugate
在20mM His-乙酸盐(pH 5.5)、50mM NaCl(等度梯度)中，在Sephacryl S-300HR(GE Healthcare)柱上使用SEC纯化实施例8中制备的Cys修饰的AFD.v14-HGEO-400MA缀合物(含有HGEO-400MA PEG)(下文中称为“AFD.v14HGEO缀合物”或“AFD.v14.C+HGEO八聚体”)。如上所描述的，使用Sephacryl S-400HR确定摩尔质量、缀合效率和RH(在PBS pH7.4中以0.25mL/分钟运行)。The Cys-modified AFD.v14-HGEO- 400MA conjugate (containing HGEO-400MA PEG) (hereinafter referred to as "AFD.v14HGEO conjugate" or "AFD.v14.C+HGEO octamer"). Molar mass, conjugation efficiency and RH were determined using Sephacryl S-400HR (run at 0.25 mL/min in PBS pH 7.4) as described above.
结果显示在图17A和17B中。使用HGEO-400MA PEG(HGEO核心)制备的缀合物具有15.2nm(±4.5％)的平均RH，423.8kDa(±10.6％)的平均摩尔质量，和8.2个Fab/PEG的平均值。The results are shown in Figures 17A and 17B. use The conjugate prepared with HGEO-400MA PEG (HGEO core) had an average RH of 15.2 nm (±4.5%), an average molar mass of 423.8 kDa (±10.6%), and an average of 8.2 Fab/PEG.
在Sephacryl S-300HR柱上使用SEC纯化AFD.v14HGEO缀合物之后，备选地使用Tosoh G3000PW柱和Wyatt Technology的直列静态MALS确定缀合效率和摩尔质量(如上所描述的)。摩尔质量用于估计与每个PEG连接的AFD.v14变体的数量。来自该分析的结果显示于图18A和18B以及表11中。After purification of the AFD.v14HGEO conjugate using SEC on a Sephacryl S-300HR column, conjugation efficiency and molar mass were determined alternatively using a Tosoh G3000PW column and Wyatt Technology's in-line static MALS (as described above). Molar mass was used to estimate the number of AFD.v14 variants attached to each PEG. Results from this analysis are shown in Figures 18A and 18B and Table 11.
表11Table 11
agg＝聚集物；n/d＝未确定agg = aggregate; n/d = not determined
从表11中可以看出，Cys修饰的AFD.v14变体(AFD.v14.C)与包含HGEO核心的PEG八聚体的缀合产生了包含8个Fab/PEG的缀合物。相较于与TP核心的缀合所观察到的，与HGEO核心的缀合还产生了更多的包含5-6个Fab/PEG的缀合物。最后，相比于TP核心，与HGEO核心的缀合导致更多的聚集体以及更低的缀合效率。As can be seen in Table 11, conjugation of a Cys-modified AFD.v14 variant (AFD.v14.C) to a PEG octamer comprising a HGEO core resulted in a conjugate comprising 8 Fab/PEGs. Conjugation to the HGEO core also produced more conjugates comprising 5-6 Fab/PEG than observed for conjugation to the TP core. Finally, conjugation to the HGEO core resulted in more aggregates and lower conjugation efficiency compared to the TP core.
实施例10：AFD.v14缀合物的富集Example 10: Enrichment of AFD.v14 conjugates
增加玻璃体内制剂中Fab浓度而不显著增加制剂粘度的一种方法是增加制剂中高度Fab化的缀合物的百分比。在该实例中，将阳离子交换色谱用于富集高度Fab化的缀合物。One way to increase the concentration of Fab in the intravitreal formulation without significantly increasing the viscosity of the formulation is to increase the percentage of highly Fabylated conjugate in the formulation. In this example, cation exchange chromatography was used to enrich for highly Fabylated conjugates.
合并来自实施例9a中描述的AFD.v14.C+TP八聚体的SEC纯化的级分B4-B7(8个Fab/PEG的估计Fab化)，并且在25mM乙酸钠pH 5.0中使用SP Sepharose高效强阳离子交换树脂(GE Healthcare)进行阳离子交换色谱(CEX)，其中使用0.05％Triton X-100+0.05％Triton X-114洗涤19小时以去除内毒素，然后在50个柱体积(CV)内使用25mM乙酸钠pH 5.0加上1M NaCl在10-20％之间梯度洗脱。如上所描述的，使用Shodex OH pak SB-804HQ，使用SEC-MALS+QELS分析级分。结果显示在图19A、19B、19C和表12中。SEC purified fractions B4-B7 from the AFD.v14.C+TP octamer described in Example 9a (estimated Faby of 8 Fab/PEG) were pooled and used in 25 mM sodium acetate pH 5.0 using SP Sepharose High performance strong cation exchange resin (GE Healthcare) was subjected to cation exchange chromatography (CEX) with 0.05% Triton X-100 + 0.05% Triton X-114 washing for 19 hours to remove endotoxin, then within 50 column volumes (CV) Gradient elution between 10-20% was performed using 25 mM sodium acetate pH 5.0 plus 1 M NaCl. Fractions were analyzed using SEC-MALS+QELS using Shodex OH pak SB-804HQ as described above. The results are shown in Figures 19A, 19B, 19C and Table 12.
表12Table 12
汇集在SP Sepharose树脂上由CEX获得的含有缀合物的级分，并且使用在等度条件下使用磷酸盐缓冲盐水(PBS)(pH 7.4)、150mM NaCl以0.8mL/分钟运行的300x 8mmShodex OH pak SB-804HQ进行分析。如上所描述的确定摩尔质量和RH。MALS结果显示于图20中。Conjugate-containing fractions obtained from CEX on SP Sepharose resin were pooled and 300 x 8 mm Shodex OH was run at 0.8 mL/min using phosphate buffered saline (PBS) (pH 7.4), 150 mM NaCl under isocratic conditions. pak SB-804HQ for analysis. Molar masses and RH were determined as described above. MALS results are shown in Figure 20.
富集后，获得了使用8ARM(TP)-PEG-MAL(TP核心)制备的缀合物，其具有10.5nm(±2.5％)的平均RH，407.1kDa(±0.2％)的平均摩尔质量，和7.8个Fab/PEG的平均值。After enrichment, a conjugate prepared using 8ARM(TP)-PEG-MAL(TP core) was obtained with an average RH of 10.5 nm (±2.5%), an average molar mass of 407.1 kDa (±0.2%), and an average of 7.8 Fab/PEG.
汇集上述的TP缀合物的阳离子交换色谱纯化后获得的含有缀合物的级分(CEX池)，并且与以下级分进行比较：使用Sephacryl S-300HR(GE Healthcare)(参见实施例9a)的SEC后获得的汇集级分(S300池)，和在20mM His-乙酸盐、pH 5.5、50mM NaCl(等度梯度)中使用Sephacryl S-400HR(GE Healthcare)的SEC后获得的汇集级分(数据未显示)(S400池)。将汇集的组分进行毛细管SDS凝胶电泳(CE-SDS)，并且结果显示于图21A和21B中。The conjugate-containing fractions (CEX pool) obtained after the cation exchange chromatography purification of the TP conjugates described above were pooled and compared with the following fractions: using Sephacryl S-300HR (GE Healthcare) (see Example 9a) Pooled fractions obtained after SEC (S300 pool) and SEC using Sephacryl S-400HR (GE Healthcare) in 20 mM His-acetate, pH 5.5, 50 mM NaCl (isocratic gradient) (data not shown) (S400 pool). Pooled fractions were subjected to capillary SDS gel electrophoresis (CE-SDS), and the results are shown in Figures 21A and 21B.
从图21A和21B中可以看出，S-300和S-400树脂上的纯化得到了类似的结果。然而，使用CEX富集缀合物显著地富集了包含8个Fab/PEG的缀合物的量，而去除了较低和较高分子量污染物。As can be seen in Figures 21A and 21B, purification on S-300 and S-400 resins gave similar results. However, enrichment of conjugates using CEX significantly enriched the amount of conjugates comprising 8 Fab/PEG, while lower and higher molecular weight contaminants were removed.
实施例11：PEG核心的比较Example 11: Comparison of PEG cores
使用SEC-MALS比较实施例8中制备的缀合物的性质，包括8ARM-(TP)-PEG-MAL(含有TP核心结构)、8ARM-PEG-MAL(含有HG核心结构(JenKem))、或-DX-400MA PEG(含有丁二醇核心结构，本文中称为“AFD.v14Dx缀合物”或“AFD.v14.C+DX八聚体”)。在20mMHis-乙酸盐(pH 5.5)、50mM NaCl(等度梯度)中，在Sephacryl S-300HR(GE Healthcare)柱上使用SEC纯化HG和DX缀合物。对于TP缀合物，使用如实施例9a所述在Sephacryl S-300HR上纯化后获得的汇集级分(“CEX负载”)和如实施例10所述在CEX富集后获得的汇集级分(“TP最终”)。使用Wyatt Technology的MALS以及在等度条件下使用磷酸盐缓冲盐水(PBS)(pH 7.2)、150mM NaCl以0.8mL/分钟运行的300x 8mm Shodex OH pak SB-804Hq来确定摩尔质量和缀合效率。如上所述确定RH。结果显示在图22A、22B和表13中。The properties of the conjugates prepared in Example 8 were compared using SEC-MALS, including 8ARM-(TP)-PEG-MAL (with TP core structure), 8ARM-PEG-MAL (with HG core structure (JenKem)), or - DX-400MA PEG (containing butanediol core structure, referred to herein as "AFD.v14Dx conjugate" or "AFD.v14.C+DX octamer"). HG and DX conjugates were purified using SEC on a Sephacryl S-300HR (GE Healthcare) column in 20 mM His-acetate (pH 5.5), 50 mM NaCl (isocratic gradient). For TP conjugates, pooled fractions obtained after purification on Sephacryl S-300HR as described in Example 9a ("CEX loading") and pooled fractions obtained after CEX enrichment as described in Example 10 ( "TP Ultimate"). Molar masses and conjugation efficiencies were determined using Wyatt Technology's MALS with a 300x 8mm Shodex OH pak SB-804Hq run under isocratic conditions using Phosphate Buffered Saline (PBS), pH 7.2, 150 mM NaCl, 0.8 mL/min. Determine RH as described above. The results are shown in Figures 22A, 22B and Table 13.
表13Table 13
*数据是对于最终的TP。凝胶泳道5和11(图22A)是CEX负载。* Data are for final TP. Gel lanes 5 and 11 (Fig. 22A) are CEX loads.
使用本领域已知的方法确定多分散性，并且特别地使用可商购自WyattTechnology的Astra软件来确定多分散性。Polydispersity was determined using methods known in the art, and in particular Astra software, commercially available from Wyatt Technology, was used to determine polydispersity.
可从这些结果观察到，虽然AFD.v14DX缀合物具有低的多分散性，但是其无法提供与AFD.v14.C+TP八聚体一样高的缀合效率。It can be observed from these results that although the AFD.v14DX conjugate has a low polydispersity, it cannot provide as high a conjugation efficiency as the AFD.v14.C+TP octamer.
实施例12：AFD.v14缀合物的粘度Example 12: Viscosity of AFD.v14 Conjugates
由于低粘度对于玻璃体内施用是重要的，所以在pH 7.4磷酸盐缓冲盐水(PBS)溶液中，在不同的蛋白浓度下测量实施例8中制备的、与PEG八聚体(8ARM(HG)-PEG-MAL，来自JenKem Technology,USA；AFD.v14.C+HG八聚体)或PEG四聚体(PTE-400MA，来自NOF America Corp.)缀合的Cys-修饰的AFD.v14变体(AFD.v14.C)的粘度。使用1000s-1的剪切速率在恒温于40℃的TA Instruments锥板流变仪上进行粘度测量。结果显示在图23中。Since low viscosity is important for intravitreal administration, the protein prepared in Example 8 with PEG octamer (8ARM(HG)- PEG-MAL from JenKem Technology, USA; AFD.v14.C+HG octamer) or PEG tetramer ( Viscosity of PTE-400MA from NOF America Corp.) conjugated Cys-modified AFD.v14 variant (AFD.v14.C). Viscosity measurements were performed on a TA Instruments cone and plate rheometer thermostatted at 40 °C using a shear rate of 1000 s−1 . The results are shown in Figure 23.
从图23中可以看出，在相当的粘度下，相比于缀合至四聚体，AFD.v14变体与HGEO八聚体的缀合允许更高的蛋白浓度。As can be seen in Figure 23, at comparable viscosities, conjugation of the AFD.v14 variant to the HGEO octamer allowed higher protein concentrations than conjugation to the tetramer.
将不同蛋白浓度下的AFD.v14HGEO缀合物(AFD.v14.C+HGEO八聚体)粘度与AFD.v14.C+TP八聚体的粘度进行比较。在20mM His-Ace、50mM NaCl制剂中，在pH 6.5下，在不同蛋白浓度下测量粘度。使用1000s-1的剪切速率在恒温于20℃的TAInstruments锥板流变仪上进行粘度测量。结果显示在图24中。The viscosity of the AFD.v14HGEO conjugate (AFD.v14.C+HGEO octamer) was compared with that of the AFD.v14.C+TP octamer at different protein concentrations. Viscosity was measured at various protein concentrations in a 20 mM His-Ace, 50 mM NaCl formulation at pH 6.5. Viscosity measurements were performed on a TA Instruments Cone and Plate Rheometer thermostated at 20 °C using a shear rate of 1000 s−1 . The results are shown in Figure 24.
从图24中可以看出，在相当的蛋白浓度下，AFD.v14.C+TP八聚体具有比AFD.v14.C+HGEO八聚体更低的粘度。It can be seen from Figure 24 that AFD.v14.C+TP octamer has a lower viscosity than AFD.v14.C+HGEO octamer at comparable protein concentrations.
实施例13：AFD.v14缀合物的热稳定性Example 13: Thermal stability of AFD.v14 conjugates
为了模拟AFD.v14缀合物对可能在长效递送系统中发现的条件的暴露，将AFD.v14.C+TP八聚体(实施例8中制备)的样品为在37℃在两种不同的pH和盐条件下应激数周。特别地，缀合物在以下制剂中进行评价：To simulate the exposure of the AFD.v14 conjugate to conditions likely to be found in long-acting delivery systems, samples of the AFD.v14.C+TP octamer (prepared in Example 8) were separated at 37°C at two different pH and salt stress for several weeks. Specifically, the conjugates were evaluated in the following formulations:
制剂1：10mg/mL，PBS；和，Formulation 1: 10 mg/mL, PBS; and,
制剂2：10mg/mL，20mM组氨酸HCl，50mM NaCl，pH 6.5。Formulation 2: 10 mg/mL, 20 mM Histidine HCl, 50 mM NaCl, pH 6.5.
将PBS用作人玻璃体的pH和离子强度的模拟物。将在PBS或20mM His-乙酸盐pH6.5、50mM NaCl中以10mg/mL配制的AFD.v14-TP缀合物溶液的等分试样(100μL)通过使用0.22μmSpin-X离心管(Corning)的离心过滤进行无菌过滤，然后在37℃温育0、2、4或8周(分别为(T0、T2w、T4w或T8w)。温育通过在-70℃冷冻而终止。在解冻后，如上所描述的，使用Shodex OH pak SB-804HQ通过SEC-MALS分析样品，通过CE-SDS和biacore来评价fD结合能力，如下所描述的。图25A和25B中显示了通过CE-SDS确定的缀合物的相对峰面积作为孵育时间的函数，其表明37℃下缀合物的1％/周的减少。通过SEC-MALS观察到缀合物中相似的变化，其中游离Fab和二聚体种类增加(图26)。确定测量中不存在大于标准误差的结合能力的变化(±10％)，用于在37℃温育缀合物(图28)。即使在磷酸盐缓冲盐水(PBS)中在37℃达8周后且在pH 6.5下4周后，结合能力仍保持稳定。PBS was used as a simulant for the pH and ionic strength of the human vitreous. Aliquots (100 μL) of AFD.v14-TP conjugate solutions prepared at 10 mg/mL in PBS or 20 mM His-acetate pH 6.5, 50 mM NaCl were prepared by using 0.22 μM Centrifugal filtration of Spin-X centrifuge tubes (Corning) was sterile-filtered, followed by incubation at 37°C for 0, 2, 4, or 8 weeks (T0, T2w, T4w, or T8w, respectively). Freezing terminated. After thawing, as described above, samples were analyzed by SEC-MALS using Shodex OH pak SB-804HQ, fD binding capacity was evaluated by CE-SDS and biacore, as described below. Shown in Figures 25A and 25B The relative peak areas of the conjugates determined by CE-SDS as a function of incubation time were shown, which indicated a 1%/week reduction of the conjugates at 37°C. Similar changes were observed in the conjugates by SEC-MALS, where the free Fab and dimer species increased (Figure 26). It was determined that there was no change in binding capacity greater than the standard error (± 10%) in the measurement for incubating the conjugate at 37°C (Figure 28). Even at The binding capacity remained stable after 8 weeks at 37°C in phosphate buffered saline (PBS) and after 4 weeks at pH 6.5.
a.CE-SDS分析a. CE-SDS analysis
材料和试剂：在使用前从–70℃解冻AFD.v14.C+Tp八聚体样品。氰化钾(KCN)和3-(2-呋喃甲酰基)喹啉-2-甲醛(FQ)试剂购自Molecular Probes(Eugene,OR,USA)。磷酸二氢钠和磷酸氢二钠、二甲亚砜(DMSO)和二硫苏糖醇(DTT)以及N-乙基马来酰亚胺购自Sigma-Aldrich(St.Louis,MO,USA)。十二烷基磺酸钠(SDS)、0.1M氢氧化钠(NaOH)和0.1M盐酸(HCl)试剂购自J.T.。可替换的筛选凝胶购自Beckman Coulter,Inc.(Fullerton,CA,USA)。Materials and Reagents: Thaw AFD.v14.C+Tp octamer samples from –70°C before use. Potassium cyanide (KCN) and 3-(2-furoyl)quinoline-2-carbaldehyde (FQ) reagents were purchased from Molecular Probes (Eugene, OR, USA). Sodium dihydrogen phosphate and disodium hydrogen phosphate, dimethyl sulfoxide (DMSO) and dithiothreitol (DTT), and N-ethylmaleimide were purchased from Sigma-Aldrich (St. Louis, MO, USA) . Sodium dodecylsulfonate (SDS), 0.1M sodium hydroxide (NaOH) and 0.1M hydrochloric acid (HCl) reagents were purchased from JT. Alternative screening gels were purchased from Beckman Coulter, Inc. (Fullerton, CA, USA).
溶液：使用来自Millipore纯化系统(Billerica,MA,USA)的去离子的18.2MΩ水制备水溶液。0.1M磷酸钠、pH 6.7反应缓冲液和4％SDS的溶液通过0.2μm膜滤器(Millipore,Bedford,MA,USA)过滤并在施用前稀释。在二甲亚砜(DMSO)中制备20mM荧光FQ的储备溶液并且避光储存于-20℃。在使用前解冻等分试样并用水稀释。Solutions: Aqueous solutions were prepared using deionized 18.2 MΩ water from Millipore Purification Systems (Billerica, MA, USA). A solution of 0.1 M sodium phosphate, pH 6.7 reaction buffer and 4% SDS was filtered through a 0.2 μm membrane filter (Millipore, Bedford, MA, USA) and diluted before administration. A 20 mM stock solution of fluorescent FQ was prepared in dimethyl sulfoxide (DMSO) and stored at -20°C in the dark. Thaw aliquots and dilute with water before use.
FQ标记程序：使用NAP-5凝胶过滤柱(GE Healthcare,Piscataway,NJ,USA)将AFD.v14.C+TP八聚体(300μg)的溶液换到0.5mL磷酸钠反应缓冲液中以去除可能竞争的制剂成分。将250μL的脱盐缀合物的等分试样与30μL的溶于4％SDS中的150mM N-乙基马来酰亚胺混合，并且在70℃温育5分钟以控制变性条件下的二硫键重排(参见，例如，Michels,D.A.,Brady,L.J.,Guo,A.,Balland,A.,Anal Chem 2007,79,5963-5971)。将十微升的每份2.5mM FQ和30mM KCN试剂添加到SDS-AFD.v14溶液中，并且将最终溶液在50℃温育10分钟，然后用1％SDS稀释三倍以猝灭反应。为了减少分析，将稀释的样品的等分试样与50mM DTT在70℃温育10分钟。FQ labeling procedure: Use NAP-5 gel filtration column (GE Healthcare, Piscataway, NJ, USA) to exchange the solution of AFD.v14.C+TP octamer (300μg) into 0.5mL sodium phosphate reaction buffer to remove Potentially competing formulation ingredients. A 250 μL aliquot of the desalted conjugate was mixed with 30 μL of 150 mM N-ethylmaleimide in 4% SDS and incubated at 70 °C for 5 min to control disulfide under denaturing conditions. Bond rearrangements (see, eg, Michels, DA, Brady, LJ, Guo, A., Balland, A., Anal Chem 2007, 79, 5963-5971). Ten microliters each of 2.5 mM FQ and 30 mM KCN reagents were added to the SDS-AFD.v14 solution, and the final solution was incubated at 50°C for 10 minutes, then diluted three-fold with 1% SDS to quench the reaction. To reduce analysis, aliquots of diluted samples were incubated with 50 mM DTT at 70 °C for 10 min.
CE-SDS分析：使用包裹在40℃热控制药筒中的50μm ID的31.2cm(21cm有效长度)熔融二氧化硅毛细管(Polymicro technologies,Phoenix,AZ,USA)进行AFD.v14.C+TP八聚体样品的分离。全自动Beckman PA800+系统(Beckman Coulter,Brea,CA,USA)配备有LIF检测器，并且使用32Karat 9.1版以控制仪器。LIF检测器使用在488nm处激发的3.5mW氩离子激光器；通过600±20nm带通滤波器(Edmund Optics,Barrington,NJ,USA)收集发射。电压以负模式(反极性)施加。将样品溶液在5kV下电动引入25s并在17kV下分离。在运行之间，毛细管分别用0.1M NaOH、0.1M HCl和Beckman凝胶缓冲液洗涤5分钟、1分钟、1分钟和10分钟。(参见，例如，Michels等人,Anal Chem 2007,79,5963-5971；Michels等人,Electrophoresis 2012,33,815-826)。CE-SDS analysis: AFD.v14.C+TP octamerization was performed using a 50 μm ID 31.2 cm (21 cm effective length) fused silica capillary (Polymicro technologies, Phoenix, AZ, USA) wrapped in a thermally controlled cartridge at 40 °C Separation of body samples. A fully automated Beckman PA800+ system (Beckman Coulter, Brea, CA, USA) was equipped with a LIF detector and used 32Karat version 9.1 to control the instrument. The LIF detector used a 3.5 mW argon-ion laser excited at 488 nm; emissions were collected through a 600±20 nm bandpass filter (Edmund Optics, Barrington, NJ, USA). The voltage was applied in negative mode (reverse polarity). The sample solution was electrokineticly introduced at 5 kV for 25 s and separated at 17 kV. Between runs, the capillaries were washed with 0.1 M NaOH, 0.1 M HCl, and Beckman gel buffer for 5 minutes, 1 minute, 1 minute, and 10 minutes, respectively. (See, eg, Michels et al., Anal Chem 2007, 79, 5963-5971; Michels et al., Electrophoresis 2012, 33, 815-826).
b.结合能力b. Binding ability
以下材料购自GE Healthcare：Series S CM5传感器芯片(目录号BR-1005-30)；10X运行缓冲液(目录号BR-1006-71)：0.1M Hepes pH 7.4，1.5M NaCl，0.5％20；再生溶液(目录号BR-1003-55)：10mM Gly-HCl pH 2.1；和胺偶联试剂盒(目录号BR-1000-50)。将S系列CM5传感器芯片对接到T200仪器(GE Healthcare)中，用1X运行缓冲液引发，并且遵循制造商提供的方案用70％甘油进行标准化。传感器芯片表面使用具有制造商提供的材料和建议的方案的胺偶联试剂盒激活以用于抗原的胺偶联。人因子D(fD)通过注射用10mM乙酸钠(pH 5)稀释fD(PUR#20491,2.4mg/mL)制备的含有100μg/mL抗原的溶液进行共价固定。流速为10μL/分钟，并且使用70μL的注射体积。这在fD的约5000共振单位(RU)的多次实验中产生了典型的偶联密度。通过注射70μL 1M乙醇胺封闭未反应的胺偶联位点。使用T200评价软件的校准依赖性浓度分析程序确定抗体Fab的抗原结合活性浓度。通过以下方法制备AFD.v14.C+TP八聚体的标准曲线：将储备溶液重量稀释至5μg/mL，然后连续2倍稀释以产生2.5、1.25、0.625、0.313、0.156和0.078μg/mL的样品。通过重量稀释制备测试样品以获得约0.5、1.0或1.5μg/mL的蛋白质浓度。所有样品(200μL体积)使用1X运行缓冲液制备。将60μL等分试样以10μL/分钟的流速注射到特异性抗原表面上，传感器芯片保持在25℃并且用1X运行缓冲液引发。在样品注射接近结束时由SPR信号确定与特异性抗原结合的抗体。在每个结合周期结束时通过注射30μL的10mM Gly-HClpH 2.1来洗脱结合的抗体，以引起抗体-抗原复合物的解离。将AFD.v14.C+TP八聚体的标准曲线用于确定SPR信号与抗体浓度之间的关系，使用四参数函数来分析数据。基于观察到的SPR信号，将由标准曲线计算的参数用于计算测试样品的抗原结合浓度。通过吸光度测量确定的该浓度与蛋白质浓度的比率给出了结合分数或百分比。The following materials were purchased from GE Healthcare: Series S CM5 Sensor Chip (Cat. No. BR-1005-30); 10X Running Buffer (Cat# BR-1006-71): 0.1M Hepes pH 7.4, 1.5M NaCl, 0.5% 20; Regeneration Solution (Cat. No. BR-1003-55): 10 mM Gly-HCl pH 2.1; and Amine Coupling Kit (Cat. No. BR-1000-50). Connect the S series CM5 sensor chip to In a T200 instrument (GE Healthcare), prime with IX running buffer and normalize with 70% glycerol following the manufacturer's protocol. The sensor chip surface was activated for amine coupling of the antigen using an amine conjugation kit with materials and suggested protocols provided by the manufacturer. Human factor D (fD) was covalently immobilized by injecting a solution prepared by diluting fD (PUR#20491, 2.4 mg/mL) with 10 mM sodium acetate (pH 5) containing 100 μg/mL antigen. The flow rate was 10 μL/min and an injection volume of 70 μL was used. This yielded typical coupling densities in multiple experiments at fD of about 5000 resonance units (RU). Block unreacted amine coupling sites by injecting 70 μL of 1 M ethanolamine. use The calibration-dependent concentration analysis program of the T200 evaluation software determines the antigen-binding activity concentration of the antibody Fab. A standard curve of the AFD.v14.C+TP octamer was prepared by weight dilution of the stock solution to 5 μg/mL, followed by serial 2-fold dilutions to generate 2.5, 1.25, 0.625, 0.313, 0.156 and 0.078 μg/mL sample. Test samples were prepared by gravimetric dilution to achieve protein concentrations of approximately 0.5, 1.0 or 1.5 μg/mL. All samples (200 μL volume) were prepared using IX running buffer. A 60 μL aliquot was injected onto the specific antigen surface at a flow rate of 10 μL/min, the sensor chip was kept at 25°C and primed with IX running buffer. Antibody binding to specific antigen was determined from the SPR signal near the end of sample injection. Bound antibody was eluted at the end of each binding cycle by injecting 30 μL of 10 mM Gly-HCl pH 2.1 to cause dissociation of the antibody-antigen complex. A standard curve of AFD.v14.C+TP octamer was used to determine the relationship between SPR signal and antibody concentration, and a four-parameter function was used to analyze the data. Based on the observed SPR signal, the parameters calculated from the standard curve were used to calculate the antigen binding concentration of the test samples. The ratio of this concentration to the protein concentration, determined by absorbance measurement, gives the fraction or percentage bound.
实施例14：AFD.v14缀合物的食蟹猴PKExample 14: Cynomolgus monkey PK of AFD.v14 conjugates
在食蟹猴中进行实施例8中制备的并如实施例9a和10所述纯化的AFD.v14.C+TP八聚体的体内pK研究。PK参数由单剂量实验确定。使用未缀合的、未修饰的AFD.v14(SIESD.N103S)作为对照。动物的护理遵循Genentech Institutional动物护理和使用委员会指南。In vivo pK studies of the AFD.v14.C+TP octamer prepared in Example 8 and purified as described in Examples 9a and 10 were performed in cynomolgus monkeys. PK parameters were determined by single dose experiments. Unconjugated, unmodified AFD.v14 (SIESD.N103S) was used as a control. Animal care followed Genentech Institutional Animal Care and Use Committee guidelines.
a.研究参数a. Research parameters
将食蟹猴(28只雄性动物；2kg至4kg，并且在给药时年龄为大约2-7岁)分配至四个给药组中的一个。通过30号针(100μl剂量体积)，第1组(对照)动物(4只动物)接受5mg/眼(10mg/动物)的AFD.v14的单次双侧玻璃体内剂量。通过30号针(2次注射，每眼中50μl；100μl总剂量体积)，第2组和第3组动物(每组10只动物)分别接受基于Fab重量的1或4mg/眼(2或8mg/动物)的AFD.v14.C+TP八聚体的双侧玻璃体内剂量。在注射前，使动物镇静(10mg/kg盐酸氯胺酮，0.5mg/kg地西泮)并用局部用丙美卡因处理。然后将AFD.v14或AFD.v14.C+TP八聚体通过巩膜和角膜缘后4mm处的平坦部施用，其中使针指向晶状体后方进入中玻璃体。第4组动物(4只动物)以0.4mg/动物接受AFD.v14.C+TP八聚体的单次IV大剂量(1mL)。对于IV施用，将AFD.v14.C+TP八聚体配制为10mM琥珀酸钠、10％海藻糖和0.05％吐温-20(pH5.0)。Cynomolgus monkeys (28 male animals; 2 kg to 4 kg, and approximately 2-7 years old at the time of dosing) were assigned to one of four dosing groups. Group 1 (control) animals (4 animals) received a single bilateral intravitreal dose of 5 mg/eye (10 mg/animal) of AFD.v14 via a 30 gauge needle (100 [mu]l dose volume). Groups 2 and 3 (10 animals per group) received 1 or 4 mg/eye (2 or 8 mg/ Bilateral intravitreal dose of AFD.v14.C+TP octamer in animals). Animals were sedated (10 mg/kg ketamine hydrochloride, 0.5 mg/kg diazepam) and treated with topical proparacaine prior to injection. AFD.v14 or AFD.v14.C+TP octamer was then administered through the sclera and the plana 4 mm posterior to the limbus with the needle pointing behind the lens into the middle hyaloid. Group 4 animals (4 animals) received a single IV bolus dose (1 mL) of AFD.v14.C+TP octamer at 0.4 mg/animal. For IV administration, AFD.v14.C+TP octamer was formulated in 10 mM sodium succinate, 10% trehalose and 0.05% Tween-20 (pH 5.0).
从第1、2和3组收集眼组织。在给药后的以下时间：第1组–第1(24小时)、2、4和8天；第2组和第3组–第1(24小时)、4、8、12和20天，将来自第1组的一只动物(2只眼睛)和各自来自第2组和第3组的两只动物(4只眼睛)安乐死。安乐死后，将两只眼睛摘除，并且从两只眼睛中收集玻璃体液、房水和视网膜组织。在快速冷冻眼睛几天后使用滤纸收集完整的视网膜层。确定AFD.v14和AFD.v14.C+TP八聚体在玻璃体液和房水和视网膜组织中的浓度。Eye tissues were collected from groups 1, 2 and 3. At the following times after dosing: Group 1 – Days 1 (24 hours), 2, 4 and 8; Groups 2 and 3 – Days 1 (24 hours), 4, 8, 12 and 20, One animal (2 eyes) from Group 1 and two animals (4 eyes) each from Groups 2 and 3 were euthanized. After euthanasia, both eyes were enucleated, and vitreous humor, aqueous humor and retinal tissue were collected from both eyes. Use filter paper to collect intact retinal layers several days after snap freezing the eyes. Concentrations of AFD.v14 and AFD.v14.C+TP octamer in vitreous humor and aqueous humor and retinal tissue were determined.
通过股静脉或头静脉收集所有血液样品(约1mL)。在IVT或IV给药后的以下时间抽取样品：第1组-1小时、6小时和第1(24小时)、2、3、4、5和7天；第2组和第3组-1小时、6小时和第1(24小时)、2、4、6、8、12和20天；第4组-1小时、6小时和第1(24小时)、2、4、7、11、14、17、21、24和28天。在血液收集的一小时内，将样品在室温凝结，并且通过离心分离血清并在-60℃至-80℃储存。All blood samples (approximately 1 mL) were collected via the femoral or cephalic vein. Samples were drawn at the following times after IVT or IV administration: Group 1 - 1 hour, 6 hours and Days 1 (24 hours), 2, 3, 4, 5 and 7; Groups 2 and 3 - 1 Hours, 6 hours and 1st (24 hours), 2, 4, 6, 8, 12 and 20 days; Group 4 - 1 hour, 6 hours and 1st (24 hours), 2, 4, 7, 11, 14, 17, 21, 24 and 28 days. Within one hour of blood collection, samples were clotted at room temperature, and serum was separated by centrifugation and stored at -60°C to -80°C.
研究方案的细节在表14中列出。Details of the study protocol are listed in Table 14.
表14.食蟹猴pK研究参数Table 14. Cynomolgus Monkey pK Study Parameters
b.AFD.v14和AFD.v14.C+TP八聚体的药代动力学测定b. Pharmacokinetic determination of AFD.v14 and AFD.v14.C+TP octamer
将Gyrolab XP测定用于定量食蟹猴血清、玻璃体液、房水和视网膜匀浆中的AFD.v14和AFD.v14.C+TP八聚体。将样品在样品缓冲液(磷酸盐缓冲盐水(PBS)，0.5％牛血清白蛋白(BSA)，15ppm Proclin(Sigma-Aldrich)，0.05％吐温20，0.25％CHAPS，50μg/mLmuIgG(Equitech Bio，目录号SLM66)，5mM EDTA(pH 7.4))中以1:4–1:3000稀释。通过在样品缓冲液中连续稀释AFD.v14或AFD.v14.C+TP八聚体(从2.06-1500ng/mL)制备AFD.v14和AFD.v14.C+TP八聚体的标准曲线。以PBS/0.01％吐温20/0.02％NaN3中的100μg/mL的生物素缀合的山羊抗-人IgG(HC+LC,Bethyl，目录号A80-319B)和Rexxip F(Gyrolab)中的25nM的Alexa-抗-CDR(克隆234，Genentech)应用捕获和检测试剂测定在Gyrolab Bioaffy200CD上进行，并且洗涤步骤使用PBS/0.01％吐温20/0.02％NaN3，接着使用Gyros pH 11洗涤缓冲液。如制造商所描述的以1％PMT设置运行仪器和分析数据。AFD.v14和AFD.v14.C+TP八聚体缀合物的浓度由其标准曲线的五参数拟合确定。食蟹猴血清、玻璃体液、房水和视网膜匀浆中的AFD.v14和AFD.v14.C+TP八聚体缀合物的最小可定量浓度为8.24ng/mL(0.16nM)。The Gyrolab XP assay was used to quantify AFD.v14 and AFD.v14.C+TP octamers in cynomolgus monkey serum, vitreous humor, aqueous humor and retinal homogenates. Samples were prepared in sample buffer (phosphate-buffered saline (PBS), 0.5% bovine serum albumin (BSA), 15ppm Proclin (Sigma-Aldrich), 0.05% Tween 20, 0.25% CHAPS, 50 μg/mL muIgG (Equitech Bio, catalog number SLM66), diluted 1:4–1:3000 in 5 mM EDTA (pH 7.4)). Standard curves of AFD.v14 and AFD.v14.C+TP octamer were prepared by serially diluting AFD.v14 or AFD.v14.C+TP octamer (from 2.06-1500 ng/mL) in sample buffer. Biotin-conjugated goat anti-human IgG (HC+LC, Bethyl, catalog number A80-319B) and 25 nM in Rexxip F (Gyrolab) at 100 μg/mL in PBS/0.01% Tween 20/0.02% NaN3 The Alexa-anti-CDR (clone 234, Genentech) assay using capture and detection reagents was performed on a Gyrolab Bioaffy 200CD, and the wash step used PBS/0.01% Tween 20/0.02%NaN3 followed by Gyros pH 11 wash buffer. Run the instrument and analyze data as described by the manufacturer at the 1% PMT setting. Concentrations of AFD.v14 and AFD.v14.C+TP octamer conjugates were determined from five-parameter fits of their standard curves. The minimum quantifiable concentration of AFD.v14 and AFD.v14.C+TP octamer conjugate in cynomolgus monkey serum, vitreous humor, aqueous humor and retinal homogenate was 8.24 ng/mL (0.16 nM).
玻璃体液、房水和视网膜pK结果显示于图29A(玻璃体)和29B(玻璃体，归一化的)、图30A(房水)和30B(房水、归一化的)、和图31A(视网膜)和31B(视网膜，归一化的)和下表15-17中。The vitreous humor, aqueous humor, and retina pK results are shown in Figures 29A (vitreous) and 29B (vitreous, normalized), Figures 30A (aqueous humor) and 30B (aqueous humor, normalized), and Figure 31A (retinal ) and 31B (retina, normalized) and in Tables 15-17 below.
表15.AFD.v14对照(第1组)和AFD.v14.C+TP八聚体(第2组和第3组)的玻璃体PKTable 15. Vitreous PK of AFD.v14 Control (Group 1) and AFD.v14.C+TP Octamer (Groups 2 and 3)
*相比于对照的增加* Increase compared to control
表16.AFD.v14.C+TP八聚体的房水PKTable 16. Aqueous humor PK of AFD.v14.C+TP octamer
表17.AFD.v14.C+TP八聚体的视网膜PKTable 17. Retinal PK of AFD.v14.C+TP octamer
从表15可以看出，第2组(3.5天)和第3组(5天)的玻璃体终末半衰期比未缀合的AFD.v14对照(第1组)都要更长，并且比未缀合的lampalizumab和雷珠单抗Fab的平均半衰期(约2.34天)更长。缀合的AFD.v14第2组和第3组的平均AUC/mg-剂量(约2040)高于未缀合的lampalizumab Fab的平均AUC/mg-剂量(约1733)。基于玻璃体终末半衰期，4.0mg/眼剂量比1.0mg/眼剂量清除得更慢。从表16和17以及图30和31可以看出，与未缀合的Fab相比，第2组和第3组(缀合的AFD.v14)在房水和视网膜中也观察到更长的终末半衰期。As can be seen from Table 15, the vitreous terminal half-life of Group 2 (3.5 days) and Group 3 (5 days) was longer than that of the unconjugated AFD.v14 control (Group 1), and was longer than that of the unconjugated AFD. The mean half-life (approximately 2.34 days) of combined lampalizumab and ranibizumab Fab was longer. The average AUC/mg-dose of conjugated AFD.v14 Groups 2 and 3 (approximately 2040) was higher than that of unconjugated lampalizumab Fab (approximately 1733). Based on the terminal vitreous half-life, the 4.0 mg/eye dose was cleared more slowly than the 1.0 mg/eye dose. As can be seen from Tables 16 and 17 and Figures 30 and 31, groups 2 and 3 (conjugated AFD.v14) also observed longer terminal half-life.
第1-3组的血清pK结果在图32A和32B(归一化)中显示，并且第4组的血清pK结果在图32C中显示。The serum pK results for Groups 1-3 are shown in Figures 32A and 32B (normalized), and the serum pK results for Group 4 are shown in Figure 32C.
从图32A和32B中可以看出，第2组和第3组(AFD.v14.C+TP八聚体)的血清pK曲线彼此平行(图32A)，并且在剂量归一化后重叠(图32B)。第2组和第3组的血清AUC与剂量成比例，直到最后测量的时间点。As can be seen from Figures 32A and 32B, the serum pK curves of Groups 2 and 3 (AFD.v14.C+TP octamer) were parallel to each other (Figure 32A) and overlapped after dose normalization (Figure 32B). Serum AUC in Groups 2 and 3 was dose proportional until the last measured time point.
第4组(AFD.v14.C+TP八聚体；IV剂量)的终末半衰期为7.5天，并且清除率为15.8mL/天(5.64mL/kg/天(第4组猴子的平均重量是2.8kg))。在第21、24和28天测量时，4只第4组猴中的3只的血清浓度降至检测限以下。Group 4 (AFD.v14.C+TP octamer; IV dose) had a terminal half-life of 7.5 days and a clearance rate of 15.8 mL/day (5.64 mL/kg/day (average weight of monkeys in Group 4 was 2.8kg)). Serum concentrations in 3 of 4 Group 4 monkeys fell below the limit of detection when measured on days 21, 24 and 28.
c.食蟹猴血清中因子D的药效学测定c. Pharmacodynamic determination of factor D in cynomolgus monkey serum
将夹心ELISA用于定量食蟹猴血清、玻璃体液、房水和视网膜匀浆中的因子D(fD)。在包被缓冲液(0.05M碳酸钠，pH 9.6)中将小鼠抗-人因子D克隆4676(Genentech)稀释至1μg/mL，并且在384孔Maxisorp平板(Thermo Scientific，目录号464718)上在4℃温育过夜。将平板用PBS加0.05％吐温20洗涤，并且在用PBS加0.5％牛血清白蛋白(BSA)温育2小时期间进行封闭。该温育和所有后续温育都在温和的搅拌下在室温进行。通过在样品缓冲液(补充有500ng/mL的AFD.v14治疗剂和50μg/mL小鼠IgG的测定缓冲液)中连续稀释fD(从0.04至5ng/mL)来制备食蟹猴fD标准曲线。血清样品和对照在样品缓冲液中稀释至最低1:100。在样品缓冲液中将玻璃体液、房水和视网膜匀浆样品和对照稀释至最小1:10。然后将稀释的标准品、对照和样品在平板上温育2小时，并且使用对于AFD.Ab的生物素缀合的小鼠抗-CDRmAb(克隆242，1μg/mL)检测与平板结合的fD/AFD.Ab复合物达一小时，接着还用高灵敏度SA-HRP(3ng/mL，Pierce目录号21130)检测一小时。最终的洗涤之后，添加四甲基联苯胺(Moss，目录号TMBE-1000)并显色10-15分钟，并且用1M磷酸终止反应。使用酶标仪，采用620nm参比在450nm处读板。由标准曲线的四参数拟合确定fD的浓度。食蟹猴血清中的最小可定量浓度为3.9ng/mL(0.16nM)。食蟹猴玻璃体液、房水和视网膜匀浆中的最小可定量浓度为0.39ng/ml(0.016nM)。A sandwich ELISA was used to quantify factor D (fD) in serum, vitreous humor, aqueous humor and retinal homogenates of cynomolgus monkeys. Mouse anti-human factor D clone 4676 (Genentech) was diluted to 1 μg/mL in coating buffer (0.05M sodium carbonate, pH 9.6) and plated on a 384-well Maxisorp plate (Thermo Scientific, catalog number 464718). Incubate overnight at 4°C. Plates were washed with PBS plus 0.05% Tween 20 and blocked during a 2 hour incubation with PBS plus 0.5% bovine serum albumin (BSA). This and all subsequent incubations were performed at room temperature with gentle agitation. Cynomolgus monkey fD standard curves were prepared by serially diluting fD (from 0.04 to 5 ng/mL) in sample buffer (assay buffer supplemented with 500 ng/mL of AFD.v14 therapeutic and 50 μg/mL of mouse IgG). Serum samples and controls were diluted to a minimum of 1:100 in sample buffer. Dilute vitreous humor, aqueous humor, and retinal homogenate samples and controls to a minimum of 1:10 in sample buffer. Diluted standards, controls, and samples were then incubated on the plate for 2 hours, and plate-bound fD/ AFD.Ab complexes were detected for one hour, followed by detection with High Sensitivity SA-HRP (3 ng/mL, Pierce Cat# 21130) for an additional hour. After the final wash, tetramethylbenzidine (Moss, cat# TMBE-1000) was added and developed for 10-15 minutes, and the reaction was stopped with 1M phosphoric acid. Using a microplate reader, read the plate at 450nm with a 620nm reference. Concentrations of fD were determined from a four-parameter fit of a standard curve. The minimum quantifiable concentration in cynomolgus monkey serum is 3.9ng/mL (0.16nM). The minimum quantifiable concentration in cynomolgus monkey vitreous humor, aqueous humor and retinal homogenate is 0.39ng/ml (0.016nM).
第2、3和4组的平均血清fD和AFD.v14.C+TP八聚体浓度在图33A中显示。从图33A中可以看出，在测试的所有时间点，血清fD浓度高于AFD.Ab浓度。这些结果表明所有组中均维持系统性AP补体活性。Mean serum fD and AFD.v14.C+TP octamer concentrations for Groups 2, 3 and 4 are shown in Figure 33A. As can be seen from Figure 33A, serum fD concentrations were higher than AFD.Ab concentrations at all time points tested. These results indicated that systemic AP complement activity was maintained in all groups.
第2和3组的平均眼部fD和AFD.v14.C+TP八聚体浓度在图33B中显示。从图33B中可以看出，在测试的所有时间点，玻璃体液、房水和视网膜匀浆中的AFD.Ab浓度超过了fD浓度。Mean ocular fD and AFD.v14.C+TP octamer concentrations for Groups 2 and 3 are shown in Figure 33B. As can be seen in Figure 33B, AFD.Ab concentrations in vitreous humor, aqueous humor, and retinal homogenates exceeded fD concentrations at all time points tested.
实施例15：抗-因子D抗体变体和缀合物对抑制因子D的效力Example 15: Efficacy of Anti-Factor D Antibody Variants and Conjugates for Inhibition of Factor D
在因子D依赖性因子B激活的时间分辨荧光能量转移(TR-FRET)测定中，确定包含Cys-修饰的Fab变体的AFD.Ab变体或缀合物对因子D的抑制效力。The inhibitory potency of AFD.Ab variants or conjugates comprising Cys-modified Fab variants against Factor D was determined in a Time-Resolved Fluorescence Energy Transfer (TR-FRET) assay for Factor D-dependent Factor B activation.
根据实施例8所述的程序，将Cys-修饰的AFD.v14变体(AFD.v14.C)和含有实施例7中制备的Cys-修饰的HC(SEQ ID NO:30)的AFD.v8变体(“Cys-修饰的AFD.v8变体”或“AFD.v8.C”)各自与马来酰亚胺功能化的多臂PEG四聚体(PTE-400MA，来自NOFAmerica Corp.)缀合，以形成缀合物(下文中分别称为“AFD.v14四聚体”或“AFD.v8四聚体”)。According to the procedure described in Example 8, the Cys-modified AFD.v14 variant (AFD.v14.C) and AFD.v8 containing the Cys-modified HC (SEQ ID NO:30) prepared in Example 7 The variants ("Cys-modified AFD.v8 variants" or "AFD.v8.C") were each combined with a maleimide-functionalized multi-armed PEG tetramer ( PTE-400MA from NOFA America Corp.) was conjugated to form a conjugate (hereinafter referred to as "AFD.v14 tetramer" or "AFD.v8 tetramer", respectively).
以4x浓度在酶促反应缓冲液(ERB；75mM NaCl，1mM MgCl2，25mM Tris，0.005％聚山梨醇酯20，pH 7.3)中制备AFD.Ab Fab变体、缀合物或Fab对照的稀释物，并且与0.5nM或0.2nM因子D(分别为125pM或50pM)(fD,Complement Technology；Tyler,TX)或ERB(无酶对照)以相等体积进行组合。将雷珠单抗(抗-VEGF)用作阴性对照。将因子D/AFD.Ab或因子D/缀合物混合物(7μl/孔)添加到364孔Proxiplate F plus黑色平板(Perkin Elmer HealthSciences；Waltham,MA)中，随后添加7μl/孔的底物。底物由7μg/mL(40nM)的C3b(Complement Technology)和1μg/mL(15nM)的因子B(Complement Technology)组成。将AFD.Ab Fab或缀合物、酶、辅因子和底物在温和的搅拌下在室温温育45分钟。用7μl/孔的检测试剂鸡尾酒混合物终止反应，所述混合物由8nM的生物素化的抗-因子Bb(2F12,GNEPRO282909)、4nM的铕缀合的抗-因子Ba(1C3的定制缀合，Life Technologies的GNEPRO282908；Madison,WI)和25nM的链霉抗生物素蛋白-Alexa 647组成。将平板在黑暗中在室温温育30分钟。通过在337nm处激发并检测620nm处的铕发射和665nm处的Alexa荧光发射，用PHERAstar FS酶标仪(BMG LabTech；Cary,NC)检测时间分辨荧光能量转移。通过使用四参数拟合模型(KaleidaGraph Synergy Software；Reading,PA)的非线性回归分析确定引起半最大抑制(IC50)的AFD.Ab或缀合物浓度。Dilutions of AFD.Ab Fab variants, conjugates or Fab controls were prepared at 4x concentration in Enzymatic Reaction Buffer (ERB; 75mM NaCl, 1mM MgCl2, 25mM Tris, 0.005% Polysorbate 20, pH 7.3) , and combined in equal volumes with 0.5 nM or 0.2 nM Factor D (125 pM or 50 pM, respectively) (fD, Complement Technology; Tyler, TX) or ERB (no enzyme control). Ranibizumab (anti-VEGF) was used as a negative control. Factor D/AFD.Ab or Factor D/conjugate mix (7 μl/well) was added to 364-well Proxiplate F plus black plates (Perkin Elmer HealthSciences; Waltham, MA) followed by 7 μl/well of substrate. The substrate consisted of 7 μg/mL (40 nM) of C3b (Complement Technology) and 1 μg/mL (15 nM) of Factor B (Complement Technology). The AFD.Ab Fab or conjugate, enzyme, cofactor and substrate were incubated at room temperature for 45 minutes with gentle agitation. The reaction was stopped with 7 μl/well of a detection reagent cocktail consisting of 8 nM of biotinylated anti-factor Bb (2F12, GNEPRO282909), 4 nM of europium-conjugated anti-factor Ba (a custom conjugate of 1C3, Life GNEPRO282908 from Technologies; Madison, WI) and 25 nM streptavidin-Alexa 647. Plates were incubated for 30 minutes at room temperature in the dark. Time-resolved fluorescence energy transfer was detected with a PHERAstar FS microplate reader (BMG LabTech; Cary, NC) by excitation at 337 nm and detection of europium emission at 620 nm and Alexa fluorescence emission at 665 nm. The concentration of AFD.Ab or conjugate that elicited half-maximal inhibition (IC50) was determined by nonlinear regression analysis using a four-parameter fitted model (KaleidaGraph Synergy Software; Reading, PA).
TR-FRET测定的抑制曲线显示在图34A中(表18)。Lampalizumab抑制因子D依赖性fB激活的IC50为24pM，并且IC50的标准误差为±25％。针对AFD.v8和AFD.v14的IC50与针对lampalizumab所测量的IC50相当。参见图34A(表18)。缀合的Cys-修饰的AFD.Ab格式(AFD.v8四聚体和AFD.v14四聚体)相比于未缀合的Fab的IC50差异很可能是由处理更粘稠的PEG化分子的难度所造成的(图34A，表18)。Inhibition curves for TR-FRET assays are shown in Figure 34A (Table 18). Lampalizumab inhibits Factor D-dependent fB activation with an IC50 of 24 pM, and the standard error of the IC50 is ±25%. IC50s against AFD.v8 and AFD.v14 were comparable to those measured for lampalizumab. See Figure 34A (Table 18). The difference in the IC50 of the conjugated Cys-modified AFD.Ab formats (AFD.v8 tetramer and AFD.v14 tetramer) compared to the unconjugated Fab is likely due to processing the more viscous PEGylated molecules Difficulty (Fig. 34A, Table 18).
表18.因子D依赖性因子B激活的IC50(50pM fD)Table 18. Factor D-dependent Factor B Activation IC50 (50pM fD)
在添加125pM fD的情况下，在使用上述程序的因子D依赖性因子B激活的TR-FRET测定中，还确定了AFD.v14.C+TP八聚体抑制因子D的效力。AFD.v14.C+TP八聚体(AFD.v14.C+TP八聚体)的IC50与AFD.v14、Cys-修饰的AFD.v14(“AFD.v14.C”)和lampalizumab相当。将雷珠单抗用作阴性对照。结果显示在图34B和表19中。The potency of the AFD.v14.C+TP octamer to inhibit Factor D was also determined in a TR-FRET assay of Factor D-dependent Factor B activation using the procedure described above with the addition of 125 pM fD. The IC50 of AFD.v14.C+TP octamer (AFD.v14.C+TP octamer) was comparable to AFD.v14, Cys-modified AFD.v14 (“AFD.v14.C”) and lampalizumab. Ranibizumab was used as a negative control. The results are shown in Figure 34B and Table 19.
表19.因子D依赖性因子B激活的IC50(125pM fD)Table 19. Factor D-dependent Factor B Activation IC50 (125pM fD)
*在Sephacryl S-200HR(GE Healthcare)柱上使用SEC纯化后获得的。* Obtained after purification using SEC on a Sephacryl S-200HR (GE Healthcare) column.
^在CEX富集(实施例10)后获得的。^ Obtained after CEX enrichment (Example 10).
AFD.v14TP八聚体(S300池和CEX池)的IC50是有效的，并且是与针对未缀合的Fab(lampalizumab,AFD.v14,AFD.v14.C)所测量的相当。使用阳离子交换色谱富集AFD.v14TP八聚体导致更有效的产物。The IC50 of the AFD.v14TP octamer (S300 pool and CEX pool) was potent and comparable to that measured for unconjugated Fabs (lampalizumab, AFD.v14, AFD.v14.C). Enrichment of the AFD.v14TP octamer using cation exchange chromatography resulted in a more potent product.
实施例16：抗-因子D抗体变体和缀合物对系统性补体旁路活性的影响Example 16: Effect of Anti-Factor D Antibody Variants and Conjugates on Systemic Complement Alternative Pathway Activity
先前已经显示了Lampalizumab瞬时抑制食蟹猴中的系统性补体功能(参见Loyet等人,J.Pharmacol.Exp.Ther.,2014,Vol.351,pp.527-537)。在当前实例中，在食蟹猴中评价玻璃体内施用抗-因子D抗体变体或AFD.Ab缀合物对系统性补体旁路途径(AP)活性的影响。Lampalizumab has been previously shown to transiently inhibit systemic complement function in cynomolgus monkeys (see Loyet et al., J. Pharmacol. Exp. Ther., 2014, Vol. 351, pp. 527-537). In the current example, the effect of intravitreal administration of anti-Factor D antibody variants or AFD.Ab conjugates on systemic complement alternative pathway (AP) activity was evaluated in cynomolgus monkeys.
a.食蟹猴中的药代动力学/药效学研究a. Pharmacokinetic/pharmacodynamic studies in cynomolgus monkeys
AFD.Ab变体和缀合物通过单剂量IVT或静脉内注射施用至中国来源的雄性食蟹猴(M.fascicularis)以评价分子的药代动力学(PK)和药效学(PD)。这些研究在Covance实验室(Madison,WI)进行。所有程序均遵从美国农业部动物福利法条例(9CFR 3)、实验动物护理和使用指南以及实验室动物福利办公室的规定进行。AFD.Ab variants and conjugates were administered to male cynomolgus monkeys (M. fascicularis) of Chinese origin by single dose IVT or intravenous injection to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) of the molecules. These studies were performed at Covance Laboratories (Madison, WI). All procedures were performed in compliance with the USDA Animal Welfare Act Regulations (9CFR 3), Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare.
进行了四项研究。在第一项(对照)研究中(研究1，n＝10)，将lampalizumab施用至两只眼睛，以两个50μL IVT剂量，间隔15分钟。这些动物接受10mg/眼，总计20mg/动物。在给药前(第-2天)和给药后以下时间点收集血液：45分钟，以及2、6、10、24、34、48、96、120、154、192、288和384小时。在24、48、120、192和384小时收集血液后，将每组两只动物从研究中移出，并且进行安乐死以收集眼基质。lampalizumab对照研究先前已在Loyet等人,J.Pharmacol.Exp.Ther.,2014,351:527-537中描述。Four studies were conducted. In the first (control) study (Study 1, n=10), lampalizumab was administered to both eyes in two 50 μL IVT doses 15 minutes apart. These animals received 10 mg/eye for a total of 20 mg/animal. Blood was collected predose (Day -2) and at the following time points after dosing: 45 minutes, and 2, 6, 10, 24, 34, 48, 96, 120, 154, 192, 288, and 384 hours. After blood collection at 24, 48, 120, 192 and 384 hours, two animals per group were removed from the study and euthanized to collect ocular stroma. Controlled studies of lampalizumab have been previously described in Loyet et al., J. Pharmacol. Exp. Ther., 2014, 351:527-537.
在研究2(n＝3)中，将AFD.v14施用至两只眼睛，以两个50μL IVT剂量，间隔15分钟。这些动物接受25mg/眼，总计50mg/动物。在给药前(第-1和-3天)和给药后以下时间点收集血液：30分钟，以及2、8、24、48和96小时。In Study 2 (n=3), AFD.v14 was administered to both eyes in two 50 μL IVT doses 15 minutes apart. These animals received 25 mg/eye for a total of 50 mg/animal. Blood was collected predose (days -1 and -3) and at the following time points after dosing: 30 minutes, and 2, 8, 24, 48 and 96 hours.
在研究3(n＝10)中，将AFD.v14.C+Tp八聚体施用至两只眼睛，以两个50μL IVT剂量，间隔15分钟，以提供3.9mg/眼的AFD.v14，总计7.8mg/动物的AFD.v14。在给药前(第-1和-2周)和给药后以下时间点收集血液：1、6、24、48、72、96、144、192、288和480小时。将每个时间点(在24、96、192、288和480小时)的每组两只动物从研究中移出，并且进行安乐死以收集眼基质。In Study 3 (n=10), AFD.v14.C+Tp octamer was administered to both eyes in two 50 μL IVT doses, 15 minutes apart, to provide 3.9 mg/eye of AFD.v14, total 7.8 mg/animal of AFD.v14. Blood was collected pre-dose (weeks -1 and -2) and at the following time points post-dose: 1, 6, 24, 48, 72, 96, 144, 192, 288, and 480 hours. Two animals per group at each time point (at 24, 96, 192, 288 and 480 hours) were removed from the study and euthanized to collect ocular stroma.
在研究4中，将AFD.v14.C+HG八聚体以两个50μL IVT剂量施用至两只眼睛，间隔15分钟，以提供7.1mg/眼的AFD.v14(n＝2)或11.8mg/眼的AFD.v14(n＝1)，总计14.2mg/动物的AFD.v14或23.6mg/动物的AFD.v14。在给药前(第-7和-1天)和给药后以下时间点收集血液：1、6、24、96和168小时。In Study 4, AFD.v14.C+HG octamer was administered to both eyes in two 50 μL IVT doses 15 minutes apart to provide 7.1 mg/eye of AFD.v14 (n=2) or 11.8 mg AFD.v14 per eye (n=1), totaling 14.2 mg/animal of AFD.v14 or 23.6 mg/animal of AFD.v14. Blood was collected pre-dose (days -7 and -1 ) and at the following time points post-dose: 1, 6, 24, 96 and 168 hours.
对于所有研究，通过股静脉从每只动物收集给药前和给药后血清样品以用于PK和PD分析。在每个时间点，将全血收集到血清分离管中，允许在环境温度凝结至少20分钟，然后在设置为2℃–8℃的温度范围的冷冻离心机中离心。在离心20分钟内收集血清并储存在-60℃和-80℃之间直至分析。For all studies, pre-dose and post-dose serum samples were collected from each animal via the femoral vein for PK and PD analysis. At each time point, whole blood was collected into serum separation tubes, allowed to clot at ambient temperature for at least 20 minutes, and then centrifuged in a refrigerated centrifuge set to a temperature range of 2°C–8°C. Serum was collected within 20 min of centrifugation and stored between -60°C and -80°C until analysis.
b.总AFD.v14/缀合物分析b. Total AFD.v14/conjugate analysis
将Gyrolab XP测定用于定量食蟹猴血清中的AFD.v14,AFD.v14.C+TP八聚体和AFD.v14.C+HG八聚体。将样品在样品缓冲液(磷酸盐缓冲盐水(PBS)，0.5％牛血清白蛋白(BSA)，15ppm Proclin(Sigma-Aldrich)，0.05％吐温20，0.25％CHAPS，50μg/mL muIgG(Equitech Bio，目录号SLM66)，5mM EDTA(pH 7.4))中以1:4–1:3000稀释。通过在样品缓冲液中连续稀释AFD.v14、AFD.v14.C+TP八聚体或AFD.v14.C+HG八聚体(从2.06-1500ng/mL)制备AFD.v14和AFD.v14TP和HG缀合物标准曲线。以PBS/0.01％吐温20/0.02％NaN3中的100μg/mL的生物素缀合的山羊抗-人IgG(HC+LC,Bethyl，目录号A80-319B)和Rexxip F(Gyrolab)中的25nM的Alexa-抗-CDR(克隆234，Genentech)应用捕获和检测试剂测定在Gyrolab Bioaffy 200CD上进行，并且洗涤步骤使用PBS/0.01％吐温20/0.02％NaN3，接着使用Gyros pH 11洗涤缓冲液。如制造商所描述的以1％PMT设置运行仪器和分析数据。AFD.v14、AFD.v14.C+TP八聚体和AFD.v14.C+HG八聚体的浓度由其标准曲线的五参数拟合确定。对于食蟹猴血清中的AFD.v14、AFD.v14.C+TP八聚体和AFD.v14.C+HG八聚体，最小可定量浓度为8.24ng/mL(0.16nM)。The Gyrolab XP assay was used to quantify AFD.v14, AFD.v14.C+TP octamer and AFD.v14.C+HG octamer in cynomolgus serum. Samples were prepared in sample buffer (phosphate-buffered saline (PBS), 0.5% bovine serum albumin (BSA), 15ppm Proclin (Sigma-Aldrich), 0.05% Tween 20, 0.25% CHAPS, 50 μg/mL muIgG (Equitech Bio , catalog number SLM66), diluted 1:4–1:3000 in 5 mM EDTA (pH 7.4)). AFD.v14 and AFD.v14TP were prepared by serially diluting AFD.v14, AFD.v14.C+TP octamer or AFD.v14.C+HG octamer (from 2.06-1500 ng/mL) in sample buffer and HG conjugate standard curve. Biotin-conjugated goat anti-human IgG (HC+LC, Bethyl, catalog number A80-319B) and 25 nM in Rexxip F (Gyrolab) at 100 μg/mL in PBS/0.01% Tween 20/0.02% NaN3 Alexa-anti-CDR (clone 234, Genentech) was assayed using capture and detection reagents on a Gyrolab Bioaffy 200CD, and the wash step used PBS/0.01% Tween 20/0.02% NaN3 , followed by Gyros pH 11 wash buffer . Run the instrument and analyze data as described by the manufacturer at the 1% PMT setting. Concentrations of AFD.v14, AFD.v14.C+TP octamer and AFD.v14.C+HG octamer were determined from a five-parameter fit of their standard curves. For AFD.v14, AFD.v14.C+TP octamer and AFD.v14.C+HG octamer in cynomolgus monkey serum, the minimum quantifiable concentration was 8.24ng/mL (0.16nM).
c.食蟹猴血清中因子D的药效学测定c. Pharmacodynamic determination of factor D in cynomolgus monkey serum
使用夹心ELISA来定量食蟹猴血清中的因子D(fD)。在包被缓冲液(0.05M碳酸钠，pH 9.6)中将小鼠抗-人因子D克隆4676(Genentech)稀释至1μg/mL，并且在384孔Maxisorp平板(Thermo Scientific，目录号464718)上在4℃温育过夜。将平板用PBS加0.05％吐温20洗涤，并且在用PBS加0.5％牛血清白蛋白(BSA)温育2小时期间进行封闭。该温育和所有后续温育都在温和的搅拌下在室温进行。通过在样品缓冲液(补充有500ng/mL的AFD.v14治疗剂和50μg/mL小鼠IgG的测定缓冲液)中连续稀释fD(从0.04至5ng/mL)来制备食蟹猴fD标准曲线。血清样品和对照在样品缓冲液中稀释至最低1:100。然后将稀释的标准品、对照和样品在平板上温育2小时，并且使用对于AFD.Ab的生物素缀合的小鼠抗-CDR mAb(克隆242，1μg/mL)检测与平板结合的fD/AFD.Ab复合物达一小时，接着还用高灵敏度SA-HRP(3ng/mL，Pierce目录号21130)检测一小时。最终的洗涤之后，添加四甲基联苯胺(Moss，目录号TMBE-1000)并显色10-15分钟，并且用1M磷酸终止反应。使用酶标仪，采用620nm参比在450nm处读板。由标准曲线的四参数拟合确定fD的浓度。食蟹猴血清中的最小可定量浓度为3.9ng/mL(0.16nM)。Factor D (fD) in serum of cynomolgus monkeys was quantified using a sandwich ELISA. Mouse anti-human factor D clone 4676 (Genentech) was diluted to 1 μg/mL in coating buffer (0.05M sodium carbonate, pH 9.6) and plated on a 384-well Maxisorp plate (Thermo Scientific, catalog number 464718). Incubate overnight at 4°C. Plates were washed with PBS plus 0.05% Tween 20 and blocked during a 2 hour incubation with PBS plus 0.5% bovine serum albumin (BSA). This and all subsequent incubations were performed at room temperature with gentle agitation. Cynomolgus monkey fD standard curves were prepared by serially diluting fD (from 0.04 to 5 ng/mL) in sample buffer (assay buffer supplemented with 500 ng/mL of AFD.v14 therapeutic and 50 μg/mL of mouse IgG). Serum samples and controls were diluted to a minimum of 1:100 in sample buffer. Diluted standards, controls, and samples were then incubated on the plate for 2 hours, and plate-bound fD was detected using a biotin-conjugated mouse anti-CDR mAb to AFD.Ab (clone 242, 1 μg/mL) /AFD.Ab complexes for one hour, followed by detection with High Sensitivity SA-HRP (3 ng/mL, Pierce Cat# 21130) for an additional hour. After the final wash, tetramethylbenzidine (Moss, cat# TMBE-1000) was added and developed for 10-15 minutes, and the reaction was stopped with 1M phosphoric acid. Using a microplate reader, read the plate at 450nm with a 620nm reference. Concentrations of fD were determined from a four-parameter fit of a standard curve. The minimum quantifiable concentration in cynomolgus monkey serum is 3.9ng/mL (0.16nM).
d.AP溶血测定d. AP hemolysis assay
在溶血测定中评价AFD.v14和AFD.v14.C+TP八聚体抑制AP活性的能力，其中将血清(人或猴)与兔红细胞组合，如Pangburn(Methods Enzymol,1988,162:639–653)和Katschke等人(J.Biol.Chem.,2009,284:10473–10479)所设计和描述的。为了确保补体激活不是通过经典补体途径(CP)发生的，使用C1q耗尽的人血清(Complement Technologies,Tyler,TX)，并且缓冲液包含EGTA以螯合钙(即CP活性所必需的阳离子)。The ability of AFD.v14 and AFD.v14.C+TP octamer to inhibit AP activity was evaluated in a hemolytic assay in which serum (human or monkey) was combined with rabbit erythrocytes, as in Pangburn (Methods Enzymol, 1988, 162:639– 653) and Katschke et al. (J. Biol. Chem., 2009, 284:10473-10479) designed and described. To ensure that complement activation did not occur via the classical complement pathway (CP), Clq-depleted human serum (Complement Technologies, Tyler, TX) was used and the buffer contained EGTA to chelate calcium, a cation necessary for CP activity.
将C1q耗尽的人血清用于激活AP。存在于10％C1q耗尽的人血清中的fD浓度在孔中为9.6nM，该值与先前报道的血清中的fD水平一致(Barnum等人,J.Immunol.Methods,1984,67:303–309；Loyet等人,Invest.Ophthalmol.Vis.Sci.,2012,53:6628–6637)。C1q-depleted human serum was used to activate APs. The concentration of fD present in 10% C1q-depleted human serum was 9.6 nM in the wells, a value consistent with previously reported fD levels in serum (Barnum et al., J. Immunol. Methods, 1984, 67:303– 309; Loyet et al., Invest. Ophthalmol. Vis. Sci., 2012, 53:6628–6637).
e.AFD.v14.C+HG八聚体处理的食蟹猴血清中系统性AP活性抑制的确定e. Determination of systemic inhibition of AP activity in serum of cynomolgus monkeys treated with AFD.v14.C+HG octamer
为了评价用AFD.v14.C+HG八聚体或AFD.v14.C+TP八聚体给药后系统性AP活性的任何潜在抑制的时程和剂量依赖性，进行基于平板的WIESLAB补体系统AP ELISA(来自该测定的数据在图35中称为“％AP补体活性”)或与上述体外AP溶血测定类似的离体测定(来自该测定的数据在图35中称为“％相对溶血”)。然而，在该测定中，代替将外源性AFD.v14.C+HG八聚体或AFD.v14.C+TP八聚体的稀释曲线添加到血清样品中，将样品本身连续地稀释，其具有由注射剂量的AFD.v14.C+HG八聚体或AFD.v14.C+TP八聚体引起的溶血活性的任何抑制。To evaluate the time course and dose dependence of any potential inhibition of systemic AP activity following administration with AFD.v14.C+HG octamer or AFD.v14.C+TP octamer, a plate-based WIESLAB complement system was performed AP ELISA (data from this assay is referred to as "% AP Complement Activity" in Figure 35) or an ex vivo assay similar to the in vitro AP hemolysis assay described above (data from this assay is referred to as "% Relative Hemolysis" in Figure 35) ). However, in this assay, instead of adding dilution curves of exogenous AFD.v14.C+HG octamer or AFD.v14.C+TP octamer to serum samples, the samples themselves were serially diluted, which There is no inhibition of hemolytic activity caused by injected doses of AFD.v14.C+HG octamer or AFD.v14.C+TP octamer.
如以上所描述的，制备红细胞并进行测定，用于具有以下修改的AP溶血测定。为了确定对应于最大裂解的吸光度，用无菌水(80μl/孔)制备全裂解对照，同时将GVB添加到所有其他孔(50μl)中。将食蟹猴血清样品在六个点上以1:1.5连续稀释，并且与阴性对照(仅缓冲液)一起添加到96孔U底聚丙烯板(30μl/孔)中。总裂解对照表示最大(100％)溶血。一式三份地收集数据点，并且将平均最大溶血百分比相对测定中最终血清稀释度的倒数作图。通过使用四参数拟合模型的非线性回归分析，确定50％最大溶血(AH50)值(定义为50％最大溶血)。对于没有达到饱和的那些曲线，使用曲线拟合估算AH50，其中将上渐近线固定于100％。将每个个体时间点的相对溶血百分比计算为[(个体时间点的给药后AH50)/(给药前AH50)]×100。来自每个个体正常食蟹猴的血清的AH50值可以从AH50值的整体平均值变化多达2倍。因此，将来自每个研究动物的给药前和给药后样品在相同的测定板上运行，以确保将AP活性的给药后变化与个体动物的基线补体活性直接进行比较。Red blood cells were prepared and assayed as described above for the AP hemolysis assay with the following modifications. To determine the absorbance corresponding to maximal lysis, a total lysis control was prepared with sterile water (80 μl/well), while GVB was added to all other wells (50 μl). Cynomolgus monkey serum samples were serially diluted 1 : 1.5 over six points and added to 96-well U-bottom polypropylene plates (30 μl/well) along with a negative control (buffer only). The total lysis control represents maximal (100%) hemolysis. Data points were collected in triplicate and the mean percent maximal hemolysis was plotted against the reciprocal of the final serum dilution in the assay. 50% maximal hemolysis (AH50) values (defined as 50% maximal hemolysis) were determined by non-linear regression analysis using a four parameter fitted model. For those curves that did not reach saturation, AH50 was estimated using curve fitting with the upper asymptote fixed at 100%. The relative percent hemolysis at each individual time point was calculated as [(Post-dose AH50 at individual time point)/(Pre-dose AH50)]×100. The AH50 values of serum from each individual normal cynomolgus monkey can vary by as much as 2-fold from the overall mean of the AH50 values. Therefore, pre-dose and post-dose samples from each study animal were run on the same assay plate to ensure direct comparison of post-dose changes in AP activity with baseline complement activity in individual animals.
f.结果f. Results
相比于总fD和治疗活性的相对溶血百分比显示于图35A(lampalizumab，10mg/眼)、图35B(AFD.v14，25mg/眼)和图35C(AFD.v14.C+TP八聚体，3.9mg/眼)中。lampalizumab数据(图35A)是在IVT施用10mg/眼的lampalizumab之后获得的比较数据，如Loyet等人,J.Pharmacol.Exp.Ther.,2014,351:527-537)中所描述的。从图35B可以看出，施用25mg/眼的AFD.v14瞬时抑制系统性AP活性，其中活性在施用后24小时恢复至基线，类似于先前对lampalizumab所观察到的结果(图35A)。相比之下，在施用3.9mg/眼的AFD.v14.C+TP八聚体后未观察到系统性AP抑制(图35C)。不希望受任何特定理论束缚，据信与Fab(例如，lampalizumab和AFD.v14)相比，用缀合物获得的较慢的从眼睛的清除率允许fD在较早的时间点使AFD.Ab饱和，防止系统性补体抑制。Relative percent hemolysis compared to total fD and therapeutic activity is shown in Figure 35A (lampalizumab, 10 mg/eye), Figure 35B (AFD.v14, 25 mg/eye) and Figure 35C (AFD.v14.C+TP octamer, 3.9mg/eye). The lampalizumab data (Figure 35A) are comparative data obtained after IVT administration of 10 mg/eye of lampalizumab as described in Loyet et al., J. Pharmacol. Exp. Ther., 2014, 351:527-537). As can be seen in Figure 35B, administration of 25 mg/eye of AFD.v14 transiently inhibited systemic AP activity, with activity returning to baseline by 24 hours post-administration, similar to results previously observed for lampalizumab (Figure 35A). In contrast, no systemic AP inhibition was observed after administration of 3.9 mg/eye of AFD.v14.C+TP octamer (Fig. 35C). Without wishing to be bound by any particular theory, it is believed that the slower clearance from the eye obtained with the conjugate allows fD to render AFD.Ab at an earlier time point compared to Fab (e.g., lampalizumab and AFD.v14). Saturation, preventing systemic complement inhibition.
相对于总fD和总缀合物的相对AP补体活性百分比显示于图35D(AFD.v14.C+HG八聚体，7.1mg/眼)和图35E(AFD.v14.C+HG八聚体，11.8mg/眼)中。从这些图中可以看出，对于IVT剂量高达11.8mg/眼的AFD.v14.C+HG八聚体，观察到可忽略的系统性补体抑制。由于从眼睛清除较慢，所以缀合物浓度保持低于fD的摩尔浓度，特别是在早于10小时的时间点。这与类似的AFD.Ab Fab眼部给药浓度相反，其中在这些早期时间点，摩尔浓度超过摩尔fD浓度并导致系统性AP抑制。Relative AP complement activity percentages relative to total fD and total conjugates are shown in Figure 35D (AFD.v14.C+HG octamer, 7.1 mg/eye) and Figure 35E (AFD.v14.C+HG octamer , 11.8mg/eye). As can be seen from these figures, negligible systemic complement inhibition was observed for AFD.v14.C+HG octamer at IVT doses up to 11.8 mg/eye. Due to slower clearance from the eye, the conjugate concentration remained below the molar concentration of fD, especially at time points earlier than 10 hours. This is in contrast to similar ocularly administered concentrations of AFD.Ab Fab, where at these early time points, molar concentrations exceeded molar fD concentrations and resulted in systemic AP inhibition.
本领域技术人员将认识到，或者能够仅使用常规实验确定，本文所述发明的具体实施方案的许多等价物。所述等价物旨在涵盖于以下权利要求中。Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.
尽管为了清楚理解的目的已经通过说明和实施例的方式较详细地描述了前述发明，但是描述和实施例不应被解释为限制本发明的范围。本文引用的所有专利和科学文献的公开内容明确地通过引用整体并入。Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.
上述书面说明书被认为足以使得本领域技术人员能够实施本发明。本发明的范围不受保藏的构建体的限制，因为保藏的实施方案意在作为本发明的某些方面的简单说明，并且功能相当的任何构建体都在本发明的范围内。实际上，根据以上描述，除本文中显示和描述的那些修改之外的本发明的各种修改对本领域技术人员将是明显的，并且落入所附权利要求书的范围内。The above written description is considered sufficient to enable any person skilled in the art to practice the invention. The scope of the invention is not limited by the deposited constructs, as the deposited embodiments are intended as simple illustrations of certain aspects of the invention, and any constructs that are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims.
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